Pyrimidine derivatives and methods of making and using these derivatives

ABSTRACT

This invention discloses compounds, and pharmaceutically acceptable salts thereof, useful in therapeutically and/or prophylactically treating patients with an illness. Such illnesses include cancer, and secondary infections caused by Pneumocystis carinii and Toxoplasmosis gondii in immunocompromised patients. The compounds themselves, methods of making these compounds, and methods of using these compounds are all disclosed.

The invention described herein was made in the course of work supportedin part by the National Institutes of General Medical Sciences, GrantNo. 1-RO1-GM-40998 from the National Institutes of Health, U.S.Department of Health and Human Services. The Government has certainrights in this invention.

This is a continuation-in-part application of U.S. application Ser. No.08/660,023 filed Jun. 6, 1996, which is a continuation-in-partapplication of U.S. application Ser. No. 08/515,491 filed Aug. 15, 1995,which is a divisional of U.S. application Ser. No. 08/304,044 filed Sep.12, 1994, now U.S. Pat. No. 5,508,281, which is a continuation-in-partof U.S. application Ser. No. 07/950,982 filed Sep. 23, 1992, now U.S.Pat. No. 5,346,900, which is a continuation of U.S. application Ser. No.07/829,519 filed Jan. 31, 1992, now abandoned, which in turn is acontinuation of U.S. application Ser. No. 07/682,043 filed Apr. 8, 1991,now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to pyrimidine derivative compounds andpharmaceutically acceptable salts thereof. More specifically, thisinvention relates to furo 2,3-d!pyrimidines, pyrrolo 2,3-d!pyrimidines,pyrrolo 3,2-d!pyrimidines, pyrrolo 3,4-d!pyrimidines, thieno2,3-d!pyrimidines, cyclopentapyrimidines, cyclopenta d!pyrimidines,pyrido 2,3-d!pyrimidines and pyrido 3,2-d!pyrimidines. These compoundshave been found useful in resisting and treating Pneumocystis cariniiand Toxoplasmosis gondii infections in immunocompromised patients, suchas, for example, patients with autoimmune deficiency syndrome (AIDS).These compounds are also useful as potential antitumor, antibiotic,antimalarial, antifungal or antiprotozoal agents, or as synergisticagents when used with sulfonamides and may require the use of leucovorinrescue. These compounds are also useful as antitumor agents in cancerpatients. Methods of preparing and using these compounds are alsoprovided.

2. Description of the Background Art

Various pyrimidine systems, such as the pyrido 2,3-d!pyrimidine ringsystem, have been studied due to their involvement in the inhibition ofdihydrofolate reductase (DHFR) enzymes activity. The pyrimidinederivatives disclosed herein function as DHFR inhibitors. Because DHFRreduces dihydrofolate to tetrahydrofolate, inhibition of DHFR deprivesthe cell of tetrahydrofolate, without which the cell cannot produce5,10-methylenetetrahydrofolate. 5,10-Methylenetetrahydrofolate isessential for cell growth. The inhibition of DHFR by the compounds, andpharmaceutically acceptable salts thereof, of this invention results inthe inhibition of DNA synthesis and leads to cell death. Methotrexate(MTX), trimetrexate (TMQ), piritrexim (PTX) and other folic acidanalogues function as inhibitors of cell growth by similar mechanismsinvolving the inhibition of dihydrofolate reductase.

The pyrimidine derivatives disclosed herein also function as thymidylatesynthase (TS) inhibitors. TS, along with DHFR, forms part of the systemsresponsible for the synthesis of deoxythymidylate (dTMP) fromdeoxyuridylate (dUMP). TS catalyzes the sole de novo synthesis of dTMPfrom dUMP. Inhibition of TS, therefore, deprives the cell of thymidine,which is an essential constituent of DNA. Typically, the compounds asdescribed herein where X and Y are both NH₂ or where X is NH₂ and Y is Hor CH₃ and will function as DHFR inhibitors, and compounds where X is OHand Y is NH₂, H, or CH₃ will function as TS inhibitors, although theinventor does not wish to be bound by this generality.

Drugs useful for the reduction of cancerous cells are also known.

Elslager, Edward F., et al., "Folate Antagonists. 20. Synthesis andAntitumor and Antimalarial Properties of Trimetrexate and Related 6-(Phenylamino)methyl!-2,4-quinazolinediamines" J. Med. Chem., Vol. 26 pp.1753-1760 (1983)), discloses the preparation of quinazolinediamines.This article states that the quinazolinediamines exhibit potentantimalarial, antibacterial and antitumor activity.

Methods of synthesizing diaminopyrido 2,3-d!pyrimidines having varioussubstituents are known. See Hurlbert, B. S., et al., "Studies onCondensed Pyrimidine Systems. XXIII. Synthesis of 2,4-Diaminopyrido2,3-d!pyrimidines from 6-Keto Esters", J. Med. Chem., Vol. 11, pp.703-707 (1968), and Hurlbert, B. S., and Valenti, B. F., "Studies onCondensed Pyrimidine Systems. XXIV. The Condensation of2,4,6-Triaminopyridimine with Malondialdehyde Derivatives", J. Med.Chem., Vol. 11, pp. 708-710 (1968).

Hurlbert, B. S., et al., "Studies on Condensed Pyrimidine Systems. XXV.2,4-Diaminopyrido 2,3-d!pyrimidines. Biological Data", J. Med. Chem.,Vol. 11, pp. 711-717 (1968), discloses the antimicrobial activities ofseveral subgroups of pyridopyrimidines. This article states that2,4-diaminopyrido 2,3-d!pyrimidines bearing alkyl and aralkylsubstituents in the pyrimidine moiety are inhibitors of dihydrofolatereductase having antibacterial and antiprotozoal activity and that thesecompounds potentiate sulfonamides.

Grivsky, E. M., et al., "Synthesis and Antitumor Activity of2,4-Diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido 2,3-d!pyridimine", J.Med. Chem., Vol. 23, pp. 327-329 (1980), discloses the synthesis of2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido2,3-d!pyridimine(BW301U,7). This article states that BW301U,7 is aseffective as methotrexate as an inhibitor of dihydrofolate reductasepurified from human leukemic cells and, in contrast to metoprine, hasminimal activity as an inhibitor of histamine metabolism.

Werbel, Leslie, M., et al., "Synthesis and Antimalarial Activity of aSeries of 2,4-Diamino-6- (N-alkylanilino)methyl!quinazolines 1,2!", J.Heterocyclic Chem., Vol. 24, pp. 345-349 (1987), discloses the synthesisof N6 substituted quinazoline dihydrofolate reductase inhibitors. Thisarticle states that these analogs demonstrate substantial activityagainst Plasmodium berghei infections in mice.

Piper, J. R., et al., "Syntheses and Antifolate Activity of5-Methyl-5-deaza Analogues of Aminopterin, Methotrexate, Folic Acid, andN¹⁰ -Methylfolic Acid", J. Med. Chem., Vol. 29, pp. 1080-1087 (1986),discloses that 5-methyl-5-deaza analogues of aminopterin andmethotrexate are much more growth inhibitory than methotrexate.

Pyrido 2,3-d! and 3,2-d! pyrimidines are also disclosed in U.S. Pat.Nos. 5,346,900 and 5,508,281, and co-pending application Ser. No.08/515,491, all of which are hereby expressly incorporated by reference.

Pyrrolo 2,3-d!pyrimidines are disclosed by Gangjee et al. in "Novel2,4-diamino-5-substituted-pyrrolo 2,3-d!pyrimidines As Classical andNon-Classical Antifolate Inhibitors of Dihydrofolate Reductases", J.Med. Chem., Vol. 38, pp. 2158-2165 (Jun. 6, 1995).

Gangjee, A., et al., "Classical and Non-Classical Furo 2,3-d!PyrimidinesAs Novel Antifolates: Synthesis and Biological Activities", J. Med.Chem., Vol. 37, pp. 1169-1176 (1994), discloses the furo2,3-d!pyrimidines.

In spite of the art discussed above, there remains a very real andsubstantial need for compounds that are more active and more selectivethan known compounds at resisting and treating infections caused byPneumocystis carinii and Toxoplasmosis gondii in immunocompromisedpatients, reducing the tumor size and/or the number of cancerous cellsin cancer patients, and for methods of preparing and using suchcompounds.

SUMMARY OF THE INVENTION

The present invention has met the above described need. The presentinvention provides pyrrolo 2,3-d!pyrimidine compounds, andpharmaceutically acceptable salts thereof, having the formula (1):##STR1## wherein X and Y are the same or different and are selected fromthe group consisting of OH, NH₂, H and CH₃ ;

wherein L and M are selected from the group consisting of carbon and CH,the chemical bond between L and M is selected from the group consistingof a single bond and a double bond, L and M are carbon when the bond isa double bond, and L and M are CH when the bond is a single bond;

wherein Z₂ and Z₃ are different and are selected from the groupconsisting of R₄ and ##STR2## wherein Z₂ is R₄ when Z₃ is ##STR3## andZ₂ is ##STR4## when Z₃ is R₄ ; wherein A is selected from the groupconsisting of CH and zero;

wherein B is selected from the group consisting of CH, nitrogen, N--CH₂,CH₂ --N, CH₂ --CH₂, oxygen, sulfur, sulfoxide, sulfone and zero;

wherein R₁ is selected from the group consisting of hydrogen, a loweralkyl group, a nitroso group, a formyl group and zero and R₁ is zerowhen B is zero, oxygen, sulfur, sulfoxide or sulfone;

wherein R₃ is selected from the group consisting of hydrogen, a loweralkyl group and zero, and R₃ is zero when A is zero;

wherein R₄ is selected from the group consisting of hydrogen and a loweralkyl group;

wherein R₅ is selected from the group consisting of hydrogen and a loweralkyl group;

wherein R₈ is selected from the group consisting of naphthyl, mono-, di-and tri-substituted naphthyl, thionaphthyl, thiophenyl and hydroxyphenylwhen R₁ is hydrogen and R₄ is hydrogen;

wherein R₈ is selected from the group consisting of phenyl, mono-, di-and tri-substituted phenyl, naphthyl, mono-, di- and tri-substitutednaphthyl, pyridine and p-aroyl-L-glutamate when R₁ is a lower alkylgroup and R₄ is hydrogen;

wherein R₈ is selected from the group consisting of pyridine, phenyl,mono-, di- and tri-substituted phenyl, naphthyl, and mono-, di- andtri-substituted naphthyl and p-aroyl-L-glutamate when R₁ is zero;

wherein R₈ is selected from the group consisting of phenyl, mono-, di-and tri-substituted phenyl, naphthyl, mono-, di- and tri-substitutednaphthyl and p-aroyl-L-glutamate when R₁ is hydrogen and R₄ is a loweralkyl group; and

wherein R₈ is not p-benzoyl-L-glutamate or pyridine when X is OH, A iszero, B is sulfur, R₄ is methyl and R₅ is hydrogen, and R₈ is notp-benzoyl-L-glutamate when X is OH, A is CH, B is CH, R₄ is hydrogen andR₅ is hydrogen; and

wherein each lower alkyl group is independently selected from the groupconsisting of lower alkyl groups having from 1 to 6 carbons.

The present invention also provides methods of synthesizing pyrrolo-2,3-d!pyrimidine compounds, and pharmaceutically acceptable saltsthereof, having the formula (2): ##STR5## wherein R is selected from thegroup consisting of a lower alkyl group, a p-aroyl-L-glutamate group, anaryl group, an alkylaryl group, a substituted aryl group, a substitutedalkylaryl group, a diaryl group, a triaryl group, an alkyldiaryl group,an alicyclic hydrocarbon group, an alkyltriaryl group, a substituteddiaryl group, and a substituted triaryl group, and each substituent ofthe substituted aryl group, diaryl group, triaryl group, or thesubstituted alkylaryl group, alkyldiaryl group, alkyltriaryl group isthe same or different and is selected from the group consisting of alower alkyl group, an alkoxy, a substituted alkoxyaryloxy group and ahalogen; and

wherein each lower alkyl group is independently selected from the groupconsisting of lower alkyl groups having from about 1 to 6 carbons,comprising the steps of:

a) debrominating a pyrrole;

b) fusing the product of step a) with an amidine;

c) condensing the product of step b) with a nucleophile;

d) reducing the product of step c); and

e) purifying the compounds of step d).

The present invention also provides furo 2,3-d!pyrimidine compounds, andpharmaceutically acceptable salts thereof, having the formula (4):##STR6## wherein X and Y are the same or different and are selected fromthe group consisting of OH, NH₂, H and CH₃ ;

wherein L and M are selected from the group consisting of carbon and CH,the chemical bond between L and M is selected from the group consistingof a single bond and a double bond, L and M are carbon when the bond isa double bond, and L and M are CH when the bond is a single bond;

wherein Z and Z₁ are different and are selected from the groupconsisting of R₄ and ##STR7## where Z is R₄ when Z₁ is ##STR8## and Z is##STR9## when Z₁ is R₄ ; wherein A is selected from the group consistingof CH and zero;

wherein B is selected from the group consisting of sulfur, sulfoxide,sulfone, nitrogen, oxygen, CH, N--CH₂, CH₂ --N, CH₂ --CH₂, and zero;

wherein R₁ is selected from the group consisting of hydrogen, a loweralkyl group, a nitroso group, a formyl group and zero and R₁ is zerowhen B is zero, oxygen, sulfur, sulfoxide or sulfone;

wherein R₂ is selected from the group consisting of a lower alkyl group,p-aroyl-L-glutamate, an aryl group, an alkylaryl group, a substitutedaryl group, a substituted alkylaryl group, a diaryl group, a triarylgroup, an alkyldiaryl group, an alicyclic hydrocarbon group, analkyltriaryl group, a substituted diaryl group, and a substitutedtriaryl group, and each substituent of the substituted aryl group,diaryl group, triaryl group, or the substituted alkylaryl group,alkyldiaryl group, alkyltriaryl group is the same or different and isselected from the group consisting of a lower alkyl, an alkoxy, analkoxyaryloxy group, a halogen and zero but R₂ is not3,4,5-trimethoxyphenyl, 3,4,5-trichlorophenyl, 3,4-dichlorophenyl,2,5-dimethoxyphenyl or a p-benzoyl-L-glutamate when R₁ is hydrogen andR₄ is hydrogen, and R₂ is not p-benzoyl-L-glutamate when R₁ is methyl;

wherein R₃ is selected from the group consisting of hydrogen, a loweralkyl group, and zero and R₃ is zero when A is zero;

wherein R₄ is selected from the group consisting of hydrogen, a loweralkyl group and S-R₇ where R₇ is selected from the group consisting ofphenyl, mono-, di- and tri-substituted phenyl, naphthyl, mono-, di- andtri-substituted naphthyl and p-aroyl-L-glutamate; and

wherein each lower alkyl group is independently selected from the groupconsisting of lower alkyl groups having from 1 to 6 carbons.

The present invention also provides thieno 2,3-d!pyrimidine compounds,and pharmaceutically acceptable salts thereof, having the formula (5):##STR10## wherein X and Y are the same or different and are selectedfrom the group consisting of OH, NH₂, H and CH₃ ;

wherein L and M are selected from the group consisting of carbon and CH,the chemical bond between L and M is selected from the group consistingof a single bond and a double bond, L and M are carbon when the bond isa double bond, and L and M are CH when the bond is a single bond;

wherein Z and Z₁ are different and are selected from the groupconsisting of R₄ and ##STR11## where Z is R₄ when Z₁ is ##STR12## and Zis ##STR13## when Z₁ is R₄ ; wherein A is selected from the groupconsisting of CH and zero;

wherein B is selected from the group consisting of sulfur, sulfoxide,sulfone, nitrogen, oxygen, CH, N--CH₂, CH₂ --N, CH₂ --CH₂, and zero;

wherein R₁ is selected from the group consisting of hydrogen, a loweralkyl group, a nitroso group, a formyl group and zero and R₁ is zerowhen B is zero, oxygen, sulfur, sulfoxide or sulfone;

wherein R₂ is selected from the group consisting of a lower alkyl group,an aryl group, p-aroyl-L-glutamate, an alkylaryl group, a substitutedaryl group, a substituted alkylaryl group, a diaryl group, a triarylgroup, an alkyldiaryl group, an alicyclic hydrocarbon group, analkyltriaryl group, a substituted diaryl group, and a substitutedtriaryl group, and each substituent of the substituted aryl group,diaryl group, triaryl group, or the substituted alkylaryl group,alkyldiaryl group, alkyltriaryl group is the same or different and isselected from the group consisting of a lower alkyl group, an alkoxy, analkoxyaryloxy group, a halogen and zero;

wherein R₃ is selected from the group consisting of hydrogen, a loweralkyl group and zero, and R₃ is zero when A is zero;

wherein R₄ is selected from the group consisting of hydrogen and loweralkyl group; and

wherein each lower alkyl group is independently selected from the groupconsisting of lower alkyl groups having from about 1 to 6 carbons.

The present invention also provides pyrrolo 3,2-d!pyrimidine compounds,and pharmaceutically acceptable salts thereof, having the formula (6):##STR14## wherein X and Y are the same or different and are selectedfrom the group consisting of OH, NH₂, H and CH₃ ;

wherein L and M are selected from the group consisting of carbon and CH,the chemical bond between L and M is selected from the group consistingof a single bond and a double bond, L and M are carbon when the bond isa double bond, and L and M are CH when the bond is a single bond;

wherein Z and Z₁ are different and are selected from the groupconsisting of R₄ and ##STR15## where Z is R₄ when Z₁ is ##STR16## and Zis ##STR17## when Z₁ is R₄ ; wherein A is selected from the groupconsisting of CH and zero;

wherein B is selected from the group consisting of sulfur, sulfoxide,sulfone, nitrogen, oxygen, CH, N--CH₂, CH₂ --N, CH₂ --CH₂, and zero;

wherein R₁ is selected from the group consisting of hydrogen, a loweralkyl group, a nitroso group, a formyl group and zero, and R₁ is zerowhen B is zero, oxygen, sulfur, sulfoxide or sulfone;

wherein R₂ is selected from the group consisting of a lower alkyl group,p-aroyl-L-glutamate, an aryl group, an alkylaryl group, a substitutedaryl group, a substituted alkylaryl group, a diaryl group, a triarylgroup, an alkyldiaryl group, an alicyclic hydrocarbon group, analkyltriaryl group, a substituted diaryl group, and a substitutedtriaryl group, and each substituent of the substituted aryl group,diaryl group, triaryl group, or the substituted alkylaryl group,alkyldiaryl group, or alkyltriaryl group is the same or different and isselected from the group consisting of a lower alkyl group, an alkoxy, analkoxyaryloxy group, a halogen and zero;

wherein R₃ is selected from the group consisting of hydrogen, a loweralkyl group and zero, and R₃ is zero when A is zero;

wherein R₄ is selected from the group consisting of hydrogen and loweralkyl group;

wherein R₅ is selected from the group consisting of hydrogen and loweralkyl group; and

wherein each lower alkyl group is independently selected from the groupconsisting of lower alkyl groups having from about 1 to 6 carbons.

The present invention also provides pyrrolo 3,4-d!pyrimidine compounds,and pharmaceutically acceptable salts thereof, having the formula (7):##STR18## wherein X and Y are the same or different and are selectedfrom the group consisting of OH, NH₂, H and CH₃ ;

wherein A is selected from the group consisting of CH and zero;

wherein B is selected from the group consisting of sulfur, sulfoxide,sulfone, nitrogen, oxygen, CH, N--CH₂, CH₂ --N, CH₂ --CH₂, and zero;

wherein R₁ is selected from the group consisting of hydrogen, a loweralkyl group, a nitroso group, a formyl group and zero and R₁ is zerowhen B is zero, oxygen, sulfur, sulfoxide or sulfone;

wherein R₂ is selected from the group consisting of a lower alkyl group,p-aroyl-L-glutamate, an aryl group, an alkylaryl group, a substitutedaryl group, a substituted alkylaryl group, a diaryl group, a triarylgroup, an alkyldiaryl group, an alicyclic hydrocarbon group, analkyltriaryl group, a substituted diaryl group, and a substitutedtriaryl group, and each substituent of the substituted aryl group,diaryl group, triaryl group, or the substituted alkylaryl group,alkyldiaryl group, or alkyltriaryl group is the same or different and isselected from the group consisting of a lower alkyl group, an alkoxy, analkoxyaryloxy group, a halogen and zero;

wherein R₃ is selected from the group consisting of hydrogen, a loweralkyl group, and zero, and R₃ is zero when A is zero;

wherein R₄ is selected from the group consisting of hydrogen and loweralkyl group; and

wherein each lower alkyl group is independently selected from the groupconsisting of lower alkyl groups having from about 1 to 6 carbons.

The present invention also provides cyclopentapyrimidine and cyclopentad!pyrimidine compounds, and pharmaceutically acceptable salts thereof,having the formula (8): ##STR19## wherein X and Y are the same ordifferent and are selected from the group consisting of OH, NH₂, H andCH₃ ;

wherein L and M are selected from the group consisting of carbon and CH,the chemical bond between L and M is selected from the group consistingof a single bond and a double bond, L and M are carbon when the bond isa double bond, and L and M are CH when the bond is a single bond;

wherein Z₄, Z₅ and Z₆ are different and are selected from the groupconsisting of R₄, R₅ and ##STR20## wherein A is selected from the groupconsisting of CH, sulfur and zero; wherein B is selected from the groupconsisting of sulfur, sulfoxide, sulfone, nitrogen, oxygen, CH, N--CH₂,CH₂ --N, CH₂ --CH₂, and zero;

wherein R₁ is selected from the group consisting of hydrogen, a loweralkyl group, a nitroso group, a formyl group and zero, and R₁ is zerowhen B is zero, oxygen, sulfur, sulfoxide or sulfone;

wherein R₂ is selected from the group consisting of a lower alkyl group,p-aroyl-L-glutamate, an aryl group, an alkylaryl group, a substitutedaryl group, a substituted alkylaryl group, a diaryl group, a triarylgroup, an alkyldiaryl group, an alicyclic hydrocarbon group, analkyltriaryl group, a substituted diaryl group, and a substitutedtriaryl group, and each substituent of the substituted aryl group,diaryl group, triaryl group, or the substituted alkylaryl group,alkyldiaryl group, or alkyltriaryl group is the same or different and isselected from the group consisting of a lower alkyl group, an alkylgroup, an alkoxy, an alkoxyaryloxy group, a halogen and zero;

wherein R₃ is selected from the group consisting of hydrogen, a loweralkyl group and zero, and R₃ is zero when A is zero;

wherein R₄ is selected from the group consisting of hydrogen and a loweralkyl group;

wherein R₅ is selected from the group consisting of hydrogen and a loweralkyl group;

wherein R₄ is the same or different than R₅ ;

wherein each of said R₄, R₅ and ##STR21## substituents is used once; andwherein each lower alkyl group is independently selected from the groupconsisting of lower alkyl groups having from about 1 to 6 carbons.

The present invention is also directed to tricyclic 3,2-d! and2,3-d!pyrimidine compounds, and pharmaceutically acceptable saltsthereof, having the formula (10): ##STR22## wherein X and Y are the sameor different and are selected from the group consisting of OH, NH₂, Hand CH₃ ;

wherein L and M are selected from the group consisting of carbon and CH,the chemical bond between L and M is selected from the group consistingof a single bond and a double bond, L and M are carbon when the bond isa double bond, and L and M are CH when the bond is a single bond;

wherein R₁₁ is selected from the group consisting of a lower alkylgroup, an aryl group, p-aroyl-L-glutamate, an alkylaryl group, asubstituted aryl group, a substituted alkylaryl group, a diaryl group, atriaryl group, an alkyldiaryl group, an alicyclic hydrocarbon group, analkyltriaryl group, a substituted diaryl group, and a substitutedtriaryl group, and each substituent of the substituted aryl group,diaryl group, triaryl group, or the substituted alkylaryl group,alkyldiaryl group, alkyltriaryl group is the same or different and isselected from the group consisting of a lower alkyl group, an alkoxy, analkoxyaryloxy group, a halogen and zero;

Q₃ is selected from the group consisting of oxygen, NH, sulfur and CH₂.

The present invention is also directed to pyrido 2,3-d! and3,2-d!pyrimidine compounds and pharmaceutically acceptable salts havingthe formula (11): ##STR23## wherein X and Y may be the same or differentand are selected from the group consisting of OH, NH₂, H and CH₃ ;

wherein Z and Z₁ are different and are selected from the groupconsisting of R₄ and ##STR24## where Z is R₄ when Z₁ is ##STR25## and Zis ##STR26## when Z₁ is R₄ ; wherein Q₁ and Q₂ are the same or differentand are selected from the group consisting of CH and nitrogen;

wherein A is selected from the group consisting of nitrogen, CH, sulfurand zero;

wherein B is selected from the group consisting of sulfur, sulfoxide,sulfone, CH, oxygen, nitrogen and zero; but B is not sulfur, sulfoxide,sulfone, oxygen or nitrogen when A is sulfur;

wherein R₁ is selected from the group consisting of hydrogen, a nitrosogroup, an aldehyde, a lower alkyl group, a formyl group and zero, and R₁is zero when B is zero, oxygen, sulfur, sulfoxide or sulfone;

wherein R₂ is selected from the group consisting of a lower alkyl group,p-aroyl-L-glutamate, an aryl group, an alkylaryl group, a substitutedaryl group, a substituted alkylaryl group, a diaryl group, a triarylgroup, an alkyldiaryl group, an alicyclic hydrocarbon group, anaklyltriaryl group, a substituted diaryl group and a substituted triarylgroup, and each substituent of the substituted aryl group, diaryl group,and triaryl group is the same or different and is selected from thegroup consisting of a lower alkyl group, an alkoxy, a substitutedalkoxyaryloxy group, a halogen and zero;

wherein R₃ is selected from the group consisting of H, a lower alkyl andzero, and R₃ is zero when A is zero;

wherein R₁₂ is selected from the group consisting of hydrogen andmethyl;

but R₂ is not 2,5-dimethoxyphenyl when X is NH₂, Y is NH₂, Q₁ is CH, Q₂is N, Z₁ is H, B is CH, R₁ is H and R₁₂ is methyl; and

wherein each lower alkyl group is independently selected from the groupconsisting of lower alkyl groups having from about 1 to 6 carbons.

The present invention is also directed to pyrido 2,3-d! and 3,2-d!pyrimidine compounds and pharmaceutically acceptable salts having theformula (12): ##STR27## wherein X and Y may be the same or different andare selected from the group consisting of OH, NH₂, H and CH₃ ;

wherein Z and Z₁ are different and are selected from the groupconsisting of R₄ and ##STR28## where Z is R₄ when Z₁ is ##STR29## and Zis ##STR30## when Z₁ is R₄ ; wherein Q₁ and Q₂ are the same or differentand are selected from the group consisting of CH and nitrogen;

wherein A is selected from the group consisting of nitrogen, CH, sulfurand zero;

wherein B is selected from the group consisting of sulfur, sulfoxide,sulfone, CH, oxygen, nitrogen and zero; but B is not sulfur, sulfoxide,sulfone, oxygen or nitrogen when A is sulfur;

wherein R₁ is selected from the group consisting of hydrogen, a nitrosogroup, an aldehyde, a lower alkyl group, a formyl group and zero, and R₁is zero when B is zero, oxygen, sulfur, sulfoxide or sulfone;

wherein R₂ is selected from the group consisting of a lower alkyl group,p-aroyl-L-glutamate, an aryl group, an alkylaryl group, a substitutedaryl group, a substituted alkylaryl group, a diaryl group, a triarylgroup, an alkyldiaryl group, an alicyclic hydrocarbon group, anaklyltriaryl group, a substituted diaryl group and a substituted triarylgroup, and each substituent of the substituted aryl group, diaryl group,and triaryl group is the same or different and is selected from thegroup consisting of a lower alkyl group, an alkoxy, a substitutedalkoxyaryloxy group, a halogen and zero;

wherein R₃ is selected from the group consisting of H, a lower alkyl andzero, and R₃ is zero when A is zero;

wherein R₁₂ is selected from the group consisting of hydrogen andmethyl; and

wherein each lower alkyl group is independently selected from the groupconsisting of lower alkyl groups having from about 1 to 6 carbons.

Methods of synthesizing the above compounds are also disclosed.

As used herein, the term "pharmaceutically acceptable salts" includes,but is not limited to, acetate, formate, glucuronate, ethantate,sulfonate, or other salts known to those skilled in the art.

In formulas 1 and 4-11, when X and Y are the same or different and areselected from the group consisting of OH, NH₂, H and CH₃, the enol formof the compounds is represented. The enol form is equivalent to andincludes the keto form of the compounds.

As used herein, the term "lower alkyl group" refers to an alkyl grouphaving between 1 and 6 carbons. The number of carbons in each loweralkyl group in each of formulas 1 and 3-11 can vary. For example, withreference to Formula 4, R₁ could represent a lower alkyl group having 1carbon, R₂ could represent a lower alkyl group having 2 carbons, R₃could represent a lower alkyl group having 3 carbons, and R₄ couldrepresent a lower alkyl group having 4 carbons.

As will be understood one skilled in the art, when any of the variablesused herein equal zero, that variable is not present in a particularembodiment of the general formula. In any of the above describedformulas, when A equals zero, R₃ also equals zero, and B is either zeroor is attached directly to the carbon ring. When B is zero, R₁ alsoequals zero, and R₂ and R₈ are either zero or are attached directly toA. When both A and B are zero, R₁ and R₃ are also zero and R₂ and R₈ areeither zero or are attached directly to the carbon ring.

As used herein, the term aroyl, such as for example when used within theterm p-aroyl-L-glutamate, refers to heteroaroyl, benzoyl, napthoyl,thiophenoyl, furophenoyl, pyrroyl, and any other aroyl as that termwould be understood by one skilled in the art.

This invention also provides methods for therapeutically and/orprophylactically using the compounds, and pharmaceutically acceptablesalts thereof, described herein.

This invention provides a method of using the pyrimidine derivatives ofFormulas 1, and 4-11 described herein for therapeutic and prophylacticpurposes including employing these compounds to resist and treatsecondary infections caused by Pneumocystis carinii and Toxoplasmosisgondii or other organisms in immunocompromised patients, such as forexample patients with AIDS. The immunocompromised patient has a primaryinfection caused by a retrovirus, including for example, humanimmunodeficiency virus (HIV). In addition, this invention providesmethods of using pyrimidine derivatives as antitumor, antibiotic,antimalarial, antifungal and antiprotozoal agents and as synergisticagents with sulfonamides in such patients.

This invention also provides methods of using pyrimidine derivatives fortherapeutic purposes as antitumor agents or to otherwise destroycancerous cells in cancer patients.

It is an object of this invention to provide pyrimidine derivativecompounds, and pharmaceutically acceptable salts thereof, forsubstantially inhibiting dihydrofolate reductase enzymes.

It is an object of this invention to provide pyrimidine derivativecompounds, and pharmaceutically acceptable salts thereof, forsubstantially inhibiting thymidylate synthase enzymes.

It is an object of the present invention to provide pyrimidinederivative compounds, and pharmaceutically acceptable salts thereof,having antitumor, antibiotic, antimalarial, antifungal or antiprotozoalactivity or synergistic activity with sulfonamides.

It is a further object of this invention to provide pyrimidinederivative compounds, and pharmaceutically acceptable salts thereof,having effective activity against secondary infections, such as forexample infections caused by Pneumocystis carinii and Toxoplasmosisgondii that occur in immunocompromised patients, such as for examplepatients with AIDS.

It is another object of this invention to provide pyrimidine derivativecompounds, and pharmaceutically acceptable salts thereof, havingeffective activity against tumors and other cancerous cells, such asthose caused by cancer.

It is an object of this invention to provide a method of synthesizingvarious pyrimidine derivative compounds, and pharmaceutically acceptablesalts thereof.

It is a further object of this invention to provide methods of using ina patient a therapeutically effective amount of pyrimidine derivativecompounds, or pharmaceutically acceptable salts thereof.

It is a further object of this invention to provide methods of using ina patient a prophylactically effective amount of pyrimidine derivativecompounds, or pharmaceutically acceptable salts thereof.

These and other objects of the invention will be more fully understoodfrom the drawing and the following description of the invention and theclaims appended hereto.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram of methods of preparing2,4-diamino-5-substituted-pyrrolo 2,3-d!pyrimidines.

FIG. 2 shows compounds 1-9 as synthesized by the methods shown in FIG.1.

FIG. 3 shows a schematic diagram of the methods of preparing N- 4- N-2,4-diaminofuro 2,3-d!pyrimidin-5-yl!methyl!amino!benzoyl!-L-glutamicacid and the N-9 methyl analogue thereof.

FIG. 4 shows a schematic diagram of the methods of preparing of acompound having formula 8.

FIG. 5 shows a schematic diagram of the methods of preparing severalcompounds having formula 7.

FIG. 6 shows a schematic diagram of the methods of preparing two ofcompounds having formula 4.

FIG. 7 shows a schematic diagram of the methods of preparing two ofcompounds having formula 4.

FIG. 8 shows a schematic diagram of the methods of preparing compoundshaving formula 9.

FIG. 9 shows of compounds having formula 9 as synthesized by the methodsshown in FIG. 8.

FIG. 10 shows a schematic diagram of methods of preparing tricyclic2,3-d!pyrimidines.

FIG. 11 shows a schematic diagram of the methods of preparing pyrido3,2-d!pyrimidines.

FIG. 12 shows a schematic diagram of the methods of preparing pyrido3,2-d!pyrimidines.

FIG. 13 shows a schematic diagram of the methods of preparing pyrido2,3-d!pyrimidines.

FIG. 14 shows a schematic diagram of the methods of preparing various2,4-diaminopyrido 2,3-d!pyrimidines.

FIG. 15 shows a schematic diagram of the methods of preparing2,4-diamino-6-substituted-benzylaminopyrido 2,3-d!pyrimidines.

FIG. 16 shows a schematic diagram of the methods of preparing2,4-diamino-6-(anilinomethyl)pyrido 2,3-d!pyrimidines.

FIG. 17 shows a schematic diagram of the methods of preparing pyrido3,2-d!pyrimidines.

FIG. 18 shows a schematic diagram of the methods of preparing pyrido3,2-d!pyrimidines.

DESCRIPTION OF THE PREFERRED EMBODIMENT

As used herein, the term "patients" means members of the animal kingdomincluding but not limited to human beings.

The pyrimidine derivative compounds, and pharmaceutically acceptablesalts thereof, and methods of preparing and using the compounds of thisinvention provide for the therapeutic and prophylactic treatment ofsecondary infections caused by Pneunocystis carinii and Toxoplasmosisgondii in immunocompromised patients. The patients have a primaryinfection caused by a retrovirus including human immunodeficiency virus(HIV). As will be appreciated by one skilled in the art, embodiments ofthe compounds, and pharmaceutically acceptable salts thereof, of thepresent invention which contain benzoyl-L-glutamate groups will not beapplicable to these methods. That is because Pneumocystis carinii andToxoplasmosis gondii are not generally known to take up enough of thebenzoyl-L-glutamate forms of these compounds to be effective.

In addition, these compounds function as antitumor, antibiotic,antifungal, antimalarial and antiprotozoal agents, and as synergisticagents with sulfonamides.

In addition, the compounds of this invention provide for the therapeutictreatment of tumors, or other cancerous cells, in cancer patients. Asused herein, the term "cancer" refers to any type of cancer including,but not limited to, leukemia, lung cancer, colon cancer, CNS cancer,melanoma, ovarian cancer, renal cancer, prostate cancer, and breastcancer.

The compounds disclosed in the present invention can all be generallydescribed as antifolates. The pyrrolo 2,3-d!pyrimidine compounds, furo2,3-d!-pyrimidine compounds, pyrrolo 3,2-d! and pyrrolo 3,4-d!pyrimidinecompounds, thieno 2,3-d!pyrimidine compounds, cyclopentapyrimidinecompounds, cyclopenta d!pyrimidine compounds, pyrido 2,3-d!pyrimidinecompounds, and pyrido 3,2-d!pyrimidine compounds, and pharmaceuticallyacceptable salts thereof, of this invention inhibit dihydrofolatereductase (DHFR) enzymes. The DHFR enzymes are needed for normal cellgrowth because they reduce dihydrofolate to tetrahydrofolate.Tetrahydrofolate is a precursor of 5,10-methylenetetrahydrofolate, whichis essential for DNA replication and thus cell growth. The derivativesof the present invention inhibit dihydrofolate reductase andconsequently inhibit DNA synthesis. Inhibition of DNA synthesis resultsin cell death.

In addition, the pyrimidine derivative compounds of the presentinvention, and pharmaceutically acceptable salts thereof, inhibitthymidylate synthase (TS). TS, along with DHFR, forms part of the systemresponsible for the synthesis of deoxythymidylate (dTMP) fromdeoxyuridylate (dUMP). Inhibition of TS deprives the cell of thymidine,which is an essential component of DNA.

The pyrrolo 2,3-d!pyrimidine compounds, and pharmaceutically acceptablesalts thereof, of the present invention have the general formula (1):##STR31## wherein X and Y are the same or different and are selectedfrom the group consisting of OH, NH₂, H and CH₃ ;

wherein L and M are selected from the group consisting of carbon and CH,the chemical bond between L and M is selected from the group consistingof a single bond and a double bond, L and M are carbon when the bond isa double bond, and L and M are CH when the bond is a single bond;

Wherein Z₂ and Z₃ are different and are selected from the groupconsisting of R₄ and ##STR32## where Z₂ is R₄ when Z₃ is ##STR33## andZ₂ is ##STR34## when Z₃ is R₄ ; wherein A is selected from the groupconsisting of CH and zero;

wherein B is selected from the group consisting of CH, nitrogen, N--CH₂,CH₂ --N, CH₂ --CH₂, oxygen, sulfur, sulfoxide, sulfone and zero;

wherein R₁ is selected from the group consisting of hydrogen, a loweralkyl group, a nitroso group, a formyl group and zero and R₁ is zerowhen B is zero, oxygen, sulfur, sulfoxide or sulfone;

wherein R₃ is selected from the group consisting of hydrogen, a loweralkyl group and zero, and R₃ is zero when A is zero;

wherein R₄ is selected from the group consisting of hydrogen and a loweralkyl group;

wherein R₅ is selected from the group consisting of hydrogen and a loweralkyl group;

wherein R₈ is selected from the group consisting of naphthyl, mono-, di-and tri-substituted naphthyl, thionaphthyl, thiophenyl and hydroxyphenylwhen R₁ is hydrogen and R₄ is hydrogen;

wherein R₈ is selected from the group consisting of phenyl, mono-, di-and tri-substituted phenyl, naphthyl, mono-, di- and tri-substitutednaphthyl, pyridine and p-aroyl-L-glutamate when R₁ is a lower alkylgroup and R₄ is hydrogen;

wherein R₈ is selected from the group consisting of pyridine, phenyl,mono-, di- and tri-substituted phenyl, naphthyl, and mono-, di- andtri-substituted naphthyl and p-aroyl-L-glutamate when R₁ is zero;

wherein R₈ is selected from the group consisting of phenyl, mono-, di-and tri-substituted phenyl, naphthyl, mono-, di- and tri-substitutednaphthyl and p-aroyl-L-glutamate when R₁ is hydrogen and R₄ is a loweralkyl group; and

wherein R₈ is not p-benzoyl-L-glutamate or pyridine when X is OH, A iszero, B is sulfur, R₄ is methyl and R₅ is hydrogen, and R₈ is notp-benzoyl-L-glutamate when X is OH, A is CH, B is CH, R₄ is hydrogen andR₅ is hydrogen; and

wherein each lower alkyl group is independently selected from the groupconsisting of lower alkyl groups having from 1 to 6 carbons.

Preferred embodiments of formula 1 are further recited in Table 1.

                                      TABLE 1    __________________________________________________________________________                L--M    Compound          X  Y  Bond                    A  B  R.sub.1                             R.sub.3                                R.sub.4                                   R.sub.5                                      R.sub.8    __________________________________________________________________________    301   NH.sub.2             NH.sub.2                dbl CH N  H  H  H  H  1-naphthyl    303   NH.sub.2             NH.sub.2                dbl CH N  H  H  H  H  4-OHphenyl    304   NH.sub.2             NH.sub.2                dbl CH N  CH.sub.3                             H  H  H  2,5-                                      dimethoxy-                                      phenyl    305   NH.sub.2             NH.sub.2                dbl CH N  CH.sub.3                             H  H  H  3,4-dichloro-                                      phenyl    306   NH.sub.2             NH.sub.2                dbl CH N  CH.sub.3                             H  H  H  1-naphthyl    307   NH.sub.2             NH.sub.2                dbl CH S  --*                             H  H  H  3,4-                                      dimethoxy-                                      phenyl    308   NH.sub.2             NH.sub.2                dbl CH S  -- H  H  H  3,4-dichloro-                                      phenyl    309   NH.sub.2             NH.sub.2                dbl CH S  -- H  H  H  1-naphthyl    310   NH.sub.2             NH.sub.2                dbl CH S  -- H  H  H  2-naphthyl    312   NH.sub.2             NH.sub.2                dbl CH N  CH.sub.3                             H  H  H  p-benzoyl-L-                                      glutamate    313   OH NH.sub.2                dbl CH N  H  H  CH.sub.3                                   H  p-benzoyl-L-                                      glutamate    314   OH NH.sub.2                dbl CH N--                          H  H  CH.sub.3                                   H  p-benzoyl-L-                       CH.sub.2       glutamate    315   OH NH.sub.2                dbl CH S  -- H  CH.sub.3                                   H  4-pyridine    317   OH NH.sub.2                dbl -- S  -- -- CH.sub.3                                   H  3,4-di-                                      methoxy-                                      phenyl    318   OH NH.sub.2                dbl -- S  -- -- CH.sub.3                                   H  3,4-dichloro-                                      phenyl    319   OH NH.sub.2                dbl -- S  -- -- CH.sub.3                                   H  4-chloro-                                      phenyl    320   OH NH.sub.2                dbl -- S  -- -- CH.sub.3                                   H  4-NO.sub.2 phenyl    321   OH NH.sub.2                dbl -- S  -- -- CH.sub.3                                   H  phenyl    322   OH NH.sub.2                dbl -- S  -- -- CH.sub.3                                   H  2-naphthyl    __________________________________________________________________________     "--" indicates that the substituent is zero, that is, not present in the     particular embodiment.

The most preferred embodiments of formula 1 are identified as compounds312 and 320.

The present invention is further directed to methods of synthesizing5-substituted pyrrolo 2,3-d! pyrimidines. Synthesis of these compoundsaccording to the methods of the present invention can be accomplished byconvergent synthesis. As will be understood by one skilled in the art,convergent synthesis involves the production of an intermediate productfrom which numerous additional compounds can be made. Here, thepreferred intermediate product is 2,4-diamino-5-cyano pyrrolo2,3-d!pyrimidine.

More specifically, the present invention is directed to a method ofsynthesizing a compound, and pharmaceutically acceptable salts thereof,having the formula: ##STR35## wherein R is selected from the groupconsisting of a lower alkyl group, a p-aroyl-L-glutamate group, an arylgroup, an alkylaryl group, a substituted aryl group, a substitutedalkylaryl group, a diaryl group, a triaryl group, an alkyldiaryl group,an alicyclic hydrocarbon group, an alkyltriaryl group, a substituteddiaryl group, and a substituted triaryl group, and each substituent ofthe substituted aryl group, diaryl group, triaryl group, or thesubstituted alkylaryl group, alkyldiaryl group, alkyltriaryl group isthe same or different and is selected from the group consisting of alower alkyl group, an alkoxy, a substituted alkoxyaryloxy group and ahalogen; and

wherein each lower alkyl group is independently selected from the groupconsisting of lower alkyl groups having from about 1 to 6 carbons,comprising the steps of:

a) debrominating a pyrrole;

b) fusing the product of step a) with an amidine;

c) condensing the product of step b) with a nucleophile;

d) reducing the product of step c); and

e) purifying the compounds of step d).

Preferably, the debromination of step a) is performed in a mixture ofdimethylformamide (DMF) and methanol under hydrogenation in the presenceof a palladium catalyst. Either a 5% Pd-BaCO₃ or a 10% Pd-C catalyst canbe used; 5% Pd-BaCO₃ is preferred. Typically, the debromination stepwill be completed in about 3 hours, and the reaction performed underhydrogen at a pressure of between about 40 and 60 psi, preferably atabout 50 psi.

The fusion of step b) is preferably performed with chlorformamidinehydrochloride, and more preferably performed by heating a uniformlystirred suspension of the product of step a) and chlorformamidinehydrochloride in a liquid heat transfer media and heating to atemperature of between about 150° C. and 180° C., preferably betweenabout 160° C. and 170° C., for a period of between about 36 and 50hours, preferably about 48 hours. Any liquid heat transfer media can beused, including Dowtherm-A®, available from Dow Chemical Company.

The selective nucleophile as disclosed in condensing step c) is selectedfrom the group consisting of an aniline,diethyl(p-aminoaroyl)-L-glutamate, N-methyl anddiethyl(p-aminoaroyl)-L-glutamate. Any suitable aniline can be used,including but not limited to an aniline of the formula ##STR36## whereinR₆ is selected from the group consisting of 3',4',5'-trimethoxy,3',4'-dimethoxy, 4'-methoxy, 2',5'-dimethoxy, 2',5'-diethoxy,3',4'-dichloro, 2',3'-tetramethyl, hydrogen trimethoxy, dimethoxy andmonomethoxy groups, trihalo, dihalo and monohalo groups, trialkyl,dialkyl and monoalkyl groups and combinations of methoxy groups, halogroups and lower alkyls.

Anilines of formula 3 are preferred for use in the methods of thepresent invention.

Preferably, the condensation of step c) is performed in 70% to 80%acetic acid under hydrogenation, and in the presence of a Raney nickelcatalyst. Hydrogenation times of from about 24 to 72 hours, andhydrogenation pressures of between about 50 and 60 psi, preferably 55psi, are preferred. Under these conditions, the Schiff bases areexpected to form.

Alternatively, condensation can be accomplished by heating a mixture ofthe product of step b) with formaldehyde and Raney nickel at atemperature between about 70° and 90° C., preferably 80° C., for about 2hours.

Reduction of the Schiff bases, step d), is preferably performed bystirring a solution of the product of step c) in methanol at roomtemperature using NaCNBH as the reducing agent. In addition, 50%methanolic hydrochloric acid or glacial acetic acid can be used tomaintain the pH of the reaction mixture at about 2. The reduction stepshould take about 4 hours.

Preferably, the purification of step e) is performed by a methodselected from the group consisting of silica gel column chromatographyand dissolution of the product of step d) in methanol, filtration,evaporation of the filtrate, and trituration of the residue in anhydrousdiethylether.

When either diethyl(p-aminoaroyl)-L-glutamate or N-methyldiethyl(p-aminoaroyl)-L-glutamate are used in the condensation of stepc), an additional hydrolysis step, step f), is preferably performedfollowing step e). Preferably, the hydrolysis of step f) is accomplishedby stirring a solution of the product of step e) in a 1:1 sodiumhydroxide:methanol solution at room temperature for between about 60 and84 hours, preferably about 72 hours.

Specific embodiments of these methods are discussed in the examplesbelow.

Preferred embodiments of the compounds produced by the methods of thepresent invention are further recited in Table 2.

                  TABLE 2    ______________________________________    Compound Number                  R Group    ______________________________________    1             3,4,5-trimethoxyphenyl    2             3,4-dimethoxyphenyl    3             2,5-dimethoxyphenyl    4             4-methoxyphenyl    5             2,5-diethoxyphenyl    6             3,4-dichlorophenyl    7             2',3'-(CH).sub.4 phenyl    8             phenyl    9             p-benzoyl-L-glutamate    ______________________________________

Compounds 4, 8 and 9, as defined in the above table, are preferred fortherapeutically treating cancer patients, and compounds 4 and 8 fortherapeutically and prophylactically treating infections caused byPneumocystis carinii and Toxoplasmosis gondii.

The present invention also provides pyrrolo 2,3-d!pyrimidine compounds,and pharmaceutically acceptable salts thereof, having the formula (9):##STR37## wherein Q is selected from the group consisting of nitrogenand sulfur; wherein R is selected from the group consisting of a loweralkyl group, a p-aroyl-L-glutamate group, an aryl group, an alkylarylgroup, a substituted aryl group, a substituted alkylaryl group, a diarylgroup, a triaryl group, an alkyldiaryl group, an alicyclic hydrocarbongroup, an alkyltriaryl group, a substituted diaryl group, and asubstituted triaryl group, and each substituent of the substituted arylgroup, diaryl group, triaryl group, or the substituted alkylaryl group,alkyldiaryl group, alkyltriaryl group is the same or different and isselected from the group consisting of a lower alkyl group, an alkoxy, asubstituted alkoxyaryloxy group and a halogen;

wherein R₁₀ is selected from the group consisting of hydrogen and alower alkyl group; and

wherein R₁₀ is a lower alkyl when Q is nitrogen.

Methods of synthesizing compounds having formula 9 as described aboveare also provided. These methods comprise the steps of:

a) debrominating a pyrrole;

b) fusing the product of step a) with an amide;

c) condensing the product of step b) with a nucleophile;

d) reducing the product of step c); and

e) purifying the compounds of step d); wherein the nucleophile of stepc) is a compound having the general structure ##STR38## wherein Q isselected from the group consisting of nitrogen and sulfur; wherein R isselected from the group consisting of a lower alkyl group, ap-aroyl-L-glutamate group, an aryl group, an alkylaryl group, asubstituted aryl group, a substituted alkylaryl group, a diaryl group, atriaryl group, an alkyldiaryl group, an alicyclic hydrocarbon group, analkyltriaryl group, a substituted diaryl group, and a substitutedtriaryl group, and each substituent of the substituted aryl group,diaryl group, triaryl group, or the substituted alkylaryl group,alkyldiaryl group, alkyltriaryl group is the same or different and isselected from the group consisting of a lower alkyl group, an alkoxy, asubstituted alkoxyaryloxy group and a halogen;

wherein R₁₀ is selected from the group consisting of hydrogen and alower alkyl group; and

wherein R₁₀ is a lower alkyl when Q is nitrogen.

The present invention is also directed to furo 2,3-d!pyrimidinecompounds, and pharmaceutically acceptable salts thereof, having thefollowing general formula: ##STR39## wherein X and Y are the same ordifferent and are selected from the group consisting of OH, NH₂, H andCH₃ ;

wherein L and M are selected from the group consisting of carbon and CH,the chemical bond between L and M is selected from the group consistingof a single bond and a double bond, L and M are carbon when the bond isa double bond, and L and M are CH when the bond is a single bond;

wherein Z and Z₁ are different and are selected from the groupconsisting of R₄ and ##STR40## where Z is R₄ when Z₁ is ##STR41## and Zis ##STR42## when Z₁ is R₄ ; wherein A is selected from the groupconsisting of CH and zero;

wherein B is selected from the group consisting of sulfur, sulfoxide,sulfone, nitrogen, oxygen, CH, N--CH₂, CH₂ --N, CH₂ --CH₂, and zero;

wherein R₁ is selected from the group consisting of hydrogen, a loweralkyl group, a nitroso group, a formyl group and zero and R₁ is zerowhen B is zero, oxygen, sulfur, sulfoxide or sulfone;

wherein R₂ is selected from the group consisting of a lower alkyl group,p-aroyl-L-glutamate, an aryl group, an alkylaryl group, a substitutedaryl group, a substituted alkylaryl group, a diaryl group, a triarylgroup, an alkyldiaryl group, an alicyclic hydrocarbon group, analkyltriaryl group, a substituted diaryl group, and a substitutedtriaryl group, and each substituent of the substituted aryl group,diaryl group, triaryl group, or the substituted alkylaryl group,alkyldiaryl group, alkyltriaryl group is the same or different and isselected from the group consisting of a lower alkyl, an alkoxy, analkoxyaryloxy group, a halogen and zero but R₂ is not3,4,5-trimethoxyphenyl, 3,4,5-trichlorophenyl, 3,4-dichlorophenyl,2,5-dimethoxyphenyl or a p-benzoyl-L-glutamate when R₁ is hydrogen andR₄ is hydrogen, and R₂ is not p-benzoyl-L-glutamate when R₁ is methyl;

wherein R₃ is selected from the group consisting of hydrogen, a loweralkyl group, and zero and R₃ is zero when A is zero;

wherein R₄ is selected from the group consisting of hydrogen, a loweralkyl group and S-R₇ where R₇ is selected from the group consisting ofphenyl, mono-, di- and tri-substituted phenyl, naphthyl, mono-, di- andtri-substituted naphthyl and p-aroyl-L-glutamate; and

wherein each lower alkyl group is independently selected from the groupconsisting of lower alkyl groups having from 1 to 6 carbons.

Particularly preferred embodiments of formula 4 are recited below inTable 3. For all of the embodiments described in Table 3, X is NH₂, Y isNH₂ and the bond between L and M is a double bond.

                  TABLE 3    ______________________________________    Compound            A      B     R.sub.1                              R.sub.2     R.sub.3                                               R.sub.4    ______________________________________    159     CH     S     --   phenyl      H    H    160     CH     S     --   1-naphthyl  H    H    161     CH     S     --   2-naphthyl  H    H    162     CH     N     H    1-naphthyl  H    H    163     CH     N     H    2-naphthyl  H    H    164     CH     O     --   2-naphthyl  H    H    165     CH     N     H    2-phenoxyphenyl                                          H    H    166     CH     N     H    4-phenoxyphenyl                                          H    H    167     CH     N     H    2-phenylphenyl                                          H    H    169     CH     N     H    2',5'-dichlorophenyl                                          H    H    171     CH     N     CH.sub.3                              3',4'-dichlorophenyl                                          H    H    172     CH     N     CH.sub.3                              3',4',5'-trichlorophenyl                                          H    H    175     CH     N     H    3'-methoxyphenyl                                          H    H    177     CH     H     --   --          H    S--R.sub.7 *    178     CH     H     --   --          H    S--R.sub.7 †    179     --     --    --   phenyl      --   H    ______________________________________     *R.sub.7 equals 1naphthyl     †R.sub.7 equals 2naphthyl

Compounds 161 and 167 as described in Table 3 are most preferred fortherapeutically treating cancer patients, and therapeutically andprophylactically treating infections caused by Pneumocystis carinii andToxoplasmosis gondii.

The present invention is also directed to methods for synthesizing thecompound, and pharmaceutically acceptable salts thereof, having theformula ##STR43## wherein X and Y are the same or different and areselected from the group consisting of OH, NH₂, H and CH₃ ;

wherein L and M are selected from the group consisting of carbon and CH,the chemical bond between L and M is selected from the group consistingof a single bond and a double bond, L and M are carbon when the bond isa double bond, and L and M are CH when the bond is a single bond;

wherein Z and Z₁ are different and are selected from the groupconsisting of R₄ and ##STR44## where Z is R₄ when Z₁ is ##STR45## and Zis ##STR46## when Z₁ is R₄ ; wherein A is selected from the groupconsisting of CH and zero;

wherein B is selected from the group consisting of sulfur, sulfoxide,sulfone, nitrogen, oxygen, CH, N--CH₂, CH₂ --N, CH₂ --CH₂, and zero;

wherein R₁ is selected from the group consisting of hydrogen, a loweralkyl group, a nitroso group, a formyl group and zero and R₁ is zerowhen B is zero, oxygen, sulfur, sulfoxide or sulfone;

wherein R₂ is selected from the group consisting of a lower alkyl group,p-aroyl-L-glutamate, an aryl group, an alkylaryl group, a substitutedaryl group, a substituted alkylaryl group, a diaryl group, a triarylgroup, an alkyldiaryl group, an alicyclic hydrocarbon group, analkyltriaryl group, a substituted diaryl group, and a substitutedtriaryl group, and each substituent of the substituted aryl group,diaryl group, triaryl group, or the substituted alkylaryl group,alkyldiaryl group, alkyltriaryl group is the same or different and isselected from the group consisting of a lower alkyl group, an alkoxy, analkoxyaryloxy group, a halogen and zero, but R₂ is not3,4,5-trimethoxyphenyl, 3,4,5-trichlorophenyl, 3,4-dichlorophenyl,2,5-dimethoxyphenyl or p-benzoyl-L-glutamate when R₁ is hydrogen and R₄is hydrogen, and R₂ is not p-benzoyl-L-glutamate when R₁ is methyl;

wherein R₃ is selected from the group consisting of hydrogen, a loweralkyl group, and zero and R₃ is zero when A is zero;

wherein R₄ is selected from the group consisting of hydrogen, a loweralkyl group and S-R₇ where R₇ is selected from the group consisting ofphenyl, mono-, di- and tri-substituted phenyl, naphthyl, mono-, di- andtri-substituted naphthyl and p-aroyl-L-glutamate; and

wherein each lower alkyl group is independently selected from the groupconsisting of lower alkyl groups having from 1 to 6 carbons, comprisingthe steps of:

a) stirring a pyrimidine and a substituted acetone in a solvent;

b) purifying the product of step a);

c) performing nucleophilic displacement of the chloride in the productof step b); and

d) purifying the product of step c).

In one embodiment of the methods described above, step a) is performedby stirring one equivalent each of 2,6-diamino-4-hydroxypyrimidine and1,3-dichloroacetone in DMF at room temperature for a period of betweenabout 12 and 36 hours, preferably 24 hours.

Purification of the product of step a) can be accomplished by any meansknown in the art; column chromatography is preferred. The productresulting from the purification step b) is not generally stable for longdurations at room temperature. It is, therefore, preferred that thenucleophilic displacement of step c) be performed within about 1 hour ofcompletion of step b).

The nucleophilic displacement of step c) can be accomplished by any ofvarious compounds including those selected from the group consisting of(p-aminoaroyl)-L-glutamic acid, diethyl N-(p-methylaminoaroyl)glutamate,and a nucleophile. As used in reference to the methods for synthesizinga compound, and pharmaceutically acceptable salts thereof, havingformula (4), nucleophile includes, but is not limited to aniline,substituted analines, phenols, thiophenols and substituted phenols andthiophenols.

When step c) is performed with (p-aminoaroyl)-L-glutamic acid, thepurification of step d) is preferably performed by precipitating theproduct of step c) by diluting said product with a suitable solvent,preferably water, and separating said product from unreacted startingmaterials and impurities by any means known in the art; cellulose columnchromatography is preferred. Acidification of the product is thenpreferred.

When the nucleophilic displacement of step c) is performed with diethylN- p-methylamino(aroyl)!glutamate, the purification of step d) ispreferably accomplished by stirring the product of step c) with 1Nsodium hydroxide at room temperature for between about 12 to 36,preferably 24 hours, followed by acidification.

Alternatively, when conducting the nucleophilic displacement with(p-aminoaroyl)-L-glutamic acid, the desired product can also be obtainedby reductive methylation of the intermediate with a suitable aldehyde,preferably formaldehyde, and sodium cyanoborohydride at a pH ofapproximately 6 to 7. Purification is then accomplished by any meansknown in the art, preferably by wet cellulose column, and acidificationof the product performed.

When the nucleophilic displacement reactions are accomplished with ananiline, the product of step b) is preferably mixed with anhydrousdimethylsulfoxide and two equivalents of potassium carbonate forapproximately 60 to 84 hours, preferably 72 hours, at room temperature.Heating the reaction mixture to between about 35° and 45° C. for aperiod of between about 60 and 84 hours, preferably 72 hours, increasesthe yield of the desired product. The product is then isolated fromimpurities and other unreacted starting materials by any means known inthe art. Preferably, isolation is accomplished by adding excess water tothe reaction mixture and stirring at room temperature for a period ofapproximately 6 to 8 hours to separate the product; chromatographicpurification is then performed.

The present invention is also directed to compounds, andpharmaceutically acceptable salts thereof, having the general formula 5##STR47## wherein X and Y are the same or different and are selectedfrom the group consisting of OH, NH₂, H and CH₃ ;

wherein L and M are selected from the group consisting of carbon and CH,the chemical bond between L and M is selected from the group consistingof a single bond and a double bond, L and M are carbon when the bond isa double bond, and L and M are CH when the bond is a single bond;

wherein Z and Z₁ are different and are selected from the groupconsisting of R₄ and ##STR48## where Z is R₄ when Z₁ is ##STR49## and Zis ##STR50## when Z₁ is R₄ ; wherein A is selected from the groupconsisting of CH and zero;

wherein B is selected from the group consisting of sulfur, sulfoxide,sulfone, nitrogen, oxygen, CH, N--CH₂, CH₂ --N, CH₂ --CH₂, and zero;

wherein R₁ is selected from the group consisting of hydrogen, a loweralkyl group, a nitroso group, a formyl group and zero and R₁ is zerowhen B is zero, oxygen, sulfur, sulfoxide or sulfone;

wherein R₂ is selected from the group consisting of a lower alkyl group,an aryl group, p-aroyl-L-glutamate, an alkylaryl group, a substitutedaryl group, a substituted alkylaryl group, a diaryl group, a triarylgroup, an alkyldiaryl group, an alicyclic hydrocarbon group, analkyltriaryl group, a substituted diaryl group, and a substitutedtriaryl group, and each substituent of the substituted aryl group,diaryl group, triaryl group, or the substituted alkylaryl group,alkyldiaryl group, alkyltriaryl group is the same or different and isselected from the group consisting of a lower alkyl group, an alkoxy, analkoxyaryloxy group, a halogen and zero;

wherein R₃ is selected from the group consisting of hydrogen, a loweralkyl group and zero, and R₃ is zero when A is zero;

wherein R₄ is selected from the group consisting of hydrogen and loweralkyl group; and

wherein each lower alkyl group is independently selected from the groupconsisting of lower alkyl groups having from about 1 to 6 carbons.

The present invention is also directed to compounds, andpharmaceutically acceptable salts thereof, having the general formula:##STR51## wherein X and Y are the same or different and are selectedfrom the group consisting of OH, NH₂, H and CH₃ ;

wherein L and M are selected from the group consisting of carbon and CH,the chemical bond between L and M is selected from the group consistingof a single bond and a double bond, L and M are carbon when the bond isa double bond, and L and M are CH when the bond is a single bond;

wherein Z and Z₁ are different and are selected from the groupconsisting of R₄ and ##STR52## where Z is R₄ when Z₁ is ##STR53## and Zis ##STR54## when Z₁ is R₄ ; wherein A is selected from the groupconsisting of CH and zero;

wherein B is selected from the group consisting of sulfur, sulfoxide,sulfone, nitrogen, oxygen, CH, N--CH₂, CH₂ --N, CH₂ --CH₂, and zero;

wherein R₁ is selected from the group consisting of hydrogen, a loweralkyl group, a nitroso group, a formyl group and zero, and R₁ is zerowhen B is zero, oxygen, sulfur, sulfoxide or sulfone;

wherein R₂ is selected from the group consisting of a lower alkyl group,p-aroyl-L-glutamate, an aryl group, an alkylaryl group, a substitutedaryl group, a substituted alkylaryl group, a diaryl group, a triarylgroup, an alkyldiaryl group, an alicyclic hydrocarbon group, analkyltriaryl group, a substituted diaryl group, and a substitutedtriaryl group, and each substituent of the substituted aryl group,diaryl group, triaryl group, or the substituted alkylaryl group,alkyldiaryl group, or alkyltriaryl group is the same or different and isselected from the group consisting of a lower alkyl group, an alkoxy, analkoxyaryloxy group, a halogen and zero;

wherein R₃ is selected from the group consisting of hydrogen, a loweralkyl group and zero, and R₃ is zero when A is zero;

wherein R₄ is selected from the group consisting of hydrogen and loweralkyl group;

wherein R₅ is selected from the group consisting of hydrogen and loweralkyl group; and

wherein each lower alkyl group is independently selected from the groupconsisting of lower alkyl groups having from about 1 to 6 carbons.

In preferred embodiments of formula 6, Y and X are NH₂, the bond betweenL and M is a double bond, A is zero, B is CH, R₁ is hydrogen, R₂ isselected from the group consisting of phenyl and 3,4-dichlorophenyl, R₃is zero, R₄ is selected from the group consisting of hydrogen and loweralkyl and R₅ is selected from the group consisting of hydrogen and loweralkyl.

The present invention is also directed to compounds, andpharmaceutically acceptable salts thereof, having the general formula:##STR55## wherein X and Y are the same or different and are selectedfrom the group consisting of OH, NH₂, H and CH₃ ;

wherein A is selected from the group consisting of CH and zero;

wherein B is selected from the group consisting of sulfur, sulfoxide,sulfone, nitrogen, oxygen, CH, N--CH₂, CH₂ --N, CH₂ --CH₂, and zero;

wherein R₁ is selected from the group consisting of hydrogen, a loweralkyl group, a nitroso group, a formyl group and zero and R₁ is zerowhen B is zero, oxygen, sulfur, sulfoxide or sulfone;

wherein R₂ is selected from the group consisting of a lower alkyl group,p-aroyl-L-glutamate, an aryl group, an alkylaryl group, a substitutedaryl group, a substituted alkylaryl group, a diaryl group, a triarylgroup, an alkyldiaryl group, an alicyclic hydrocarbon group, analkyltriaryl group, a substituted diaryl group, and a substitutedtriaryl group, and each substituent of the substituted aryl group,diaryl group, triaryl group, or the substituted alkylaryl group,alkyldiaryl group, or alkyltriaryl group is the same or different and isselected from the group consisting of a lower alkyl group, an alkoxy, analkoxyaryloxy group, a halogen and zero;

wherein R₃ is selected from the group consisting of hydrogen, a loweralkyl group, and zero, and R₃ is zero when A is zero;

wherein R₄ is selected from the group consisting of hydrogen and loweralkyl group; and

wherein each lower alkyl group is independently selected from the groupconsisting of lower alkyl groups having from about 1 to 6 carbons.

In preferred embodiments of formula 7, X and Y are NH₂, A is zero, B isCH, R₁ is hydrogen, R₂ is selected from the group consisting of3,4,5-trimethoxybenzyl, 3,5-dimethoxybenzyl, 2,5-dimethoxybenzyl,3,4-dichlorobenzyl, 2,6-dichlorobenzyl, 2,4-dichlorobenzyl, 2-CH₂-naphthyl, C₄ H₄ benzyl and 4-benzyl-L-glutamate, R₃ is zero, R₄ ishydrogen, and R₅ is selected from the group consisting of hydrogen andmethyl. The most preferred embodiments of formula (7) are illustrated inFIG. 5.

The present invention is also directed to compounds, andpharmaceutically acceptable salts thereof, having the formula: ##STR56##wherein X and Y are the same or different and are selected from thegroup consisting of OH, NH₂, H and CH₃ ;

wherein L and M are selected from the group consisting of carbon and CH,the chemical bond between L and M is selected from the group consistingof a single bond and a double bond, L and M are carbon when the bond isa double bond, and L and M are CH when the bond is a single bond;

wherein Z₄, Z₅ and Z₆ are different and are selected from the groupconsisting of R₄, R₅ and ##STR57## wherein A is selected from the groupconsisting of CH, sulfur and zero; wherein B is selected from the groupconsisting of sulfur, sulfoxide, sulfone, nitrogen, oxygen, CH, N--CH₂,CH₂ --N, CH₂ --CH₂, and zero;

wherein R₁ is selected from the group consisting of hydrogen, a loweralkyl group, a nitroso group, a formyl group and zero, and R₁ is zerowhen B is zero, oxygen, sulfur, sulfoxide or sulfone;

wherein R₂ is selected from the group consisting of a lower alkyl group,p-aroyl-L-glutamate, an aryl group, an alkylaryl group, a substitutedaryl group, a substituted alkylaryl group, a diaryl group, a triarylgroup, an alkyldiaryl group, an alicyclic hydrocarbon group, analkyltriaryl group, a substituted diaryl group, and a substitutedtriaryl group, and each substituent of the substituted aryl group,diaryl group, triaryl group, or the substituted alkylaryl group,alkyldiaryl group, or alkyltriaryl group is the same or different and isselected from the group consisting of a lower alkyl group, an alkylgroup, an alkoxy, an alkoxyaryloxy group, a halogen and zero;

wherein R₃ is selected from the group consisting of hydrogen, a loweralkyl group and zero, and R₃ is zero when A is zero;

wherein R₄ is selected from the group consisting of hydrogen and a loweralkyl group;

wherein R₅ is selected from the group consisting of hydrogen and a loweralkyl group;

where R₄ is the same or different than R₅ ;

wherein each of said R₄, R₅ and ##STR58## substituents is used once; andwherein each lower alkyl group is independently selected from the groupconsisting of lower alkyl groups having from about 1 to 6 carbons.

In a preferred embodiment of formula 8, X and Y are NH₂, the bondbetween L and M is single, A is sulfur, B is carbon, R₁ is hydrogen, R₂is phenyl and R₃ is zero.

The present invention is also directed to tricyclic 3,2-d! and2,3-d!pyrimidine compounds, and pharmaceutically acceptable saltsthereof, having the formula (10): ##STR59## wherein X and Y are the sameor different and are selected from the group consisting of OH, NH₂, Hand CH₃ ;

wherein L and M are selected from the group consisting of carbon and CH,the chemical bond between L and M is selected from the group consistingof a single bond and a double bond, L and M are carbon when the bond isa double bond, and L and M are CH when the bond is a single bond;

wherein R₁₁ is selected from the group consisting of a lower alkylgroup, an aryl group, p-aroyl-L-glutamate, an alkylaryl group, asubstituted aryl group, a substituted alkylaryl group, a diaryl group, atriaryl group, an alkyldiaryl group, an alicyclic hydrocarbon group, analkyltriaryl group, a substituted diaryl group, and a substitutedtriaryl group, and each substituent of the substituted aryl group,diaryl group, triaryl group, or the substituted alkylaryl group,alkyldiaryl group, alkyltriaryl group is the same or different and isselected from the group consisting of a lower alkyl group, an alkoxy, analkoxyaryloxy group, a halogen and zero;

Q₃ is selected from the group consisting of oxygen, NH, sulfur and CH₂.

In a preferred embodiment of formula 10, X and Y are NH₂, the bondbetween L and M is double, Q₃ is oxygen, and R is selected from thegroup consisting of phenyl, 3,4,5-trimethoxyphenyl, 3,5-dimethoxyphenyl,2,4-dichlorophenyl, 3,4-dichlorophenyl, 2,6-dichlorophenyl andp-benzoyl-L-glutamate.

The present invention is also directed to a method for synthesizing thetricyclic pyrimidine compounds generally represented by formula (10).These methods generally include the steps of condensing abiselectrophile derived from a piperidone with a pyrimidine, preferably2,4-diamino-6-hydroxy pyrimidine. Any compatible piperidone can be used;the piperdine chosen will depend on the final pyrimidine compounddesired.

In a preferred method, the piperidone is 4-piperidone hydrochloride;protection of the piperidone is effected with ditertiarybutyl-dicarbonate in DMF at room temperature. Bromination of thisprotected piperidine is then performed in chloroform at room temperatureto yield a mixed product containing a bromopiperidine hydrobromidecompound and an N-Boc brominated compound. These compounds are thencondensed with a pyrimidine at room temperature for about 36 to 50hours, preferably 48 hours. The resulting product is then reacted withthe desired benzyl halide in anhydrous DMSO and anhydrous potassiumcarbonate for between about 48 and 72 hours at room temperature.Alternatively, the resulting product can be reacted with abenzoyl-L-glutamic acid diethyl ester instead of a benzyl halide.

The desired products of the above synthesis methods can be isolatedusing any purification means known in the art; chromatographicpurification is preferred. The isolation of the products may besimplified by adding an excess of water to the reaction mixture andstirring at room temperature for between about 1 and 3 hours, whichallows the products to separate.

The present invention is also directed to pyrido 2,3-d! and3,2-d!pyrimidine compounds and pharmaceutically acceptable salts havingthe formula (11): ##STR60## wherein X and Y may be the same or differentand are selected from the group consisting of OH, NH₂, H and CH₃ ;

wherein Z and Z₁ are different and are selected from the groupconsisting of R₄ and ##STR61## where Z is R₄ when Z₁ is ##STR62## and Zis ##STR63## when Z₁ is R₄ ; wherein Q₁ and Q₂ are the same or differentand are selected from the group consisting of CH and nitrogen;

wherein A is selected from the group consisting of nitrogen, CH, sulfurand zero;

wherein B is selected from the group consisting of sulfur, sulfoxide,sulfone, CH, oxygen, nitrogen and zero; but B is not sulfur, sulfoxide,sulfone, oxygen or nitrogen when A is sulfur;

wherein R₁ is selected from the group consisting of hydrogen, a nitrosogroup, an aldehyde, a lower alkyl group, a formyl group and zero, and R₁is zero when B is zero, oxygen, sulfur, sulfoxide or sulfone;

wherein R₂ is selected from the group consisting of a lower alkyl group,p-aroyl-L-glutamate, an aryl group, an alkylaryl group, a substitutedaryl group, a substituted alkylaryl group, a diaryl group, a triarylgroup, an alkyldiaryl group, an alicyclic hydrocarbon group, anaklyltriaryl group, a substituted diaryl group and a substituted triarylgroup, and each substituent of the substituted aryl group, diaryl group,and triaryl group is the same or different and is selected from thegroup consisting of a lower alkyl group, an alkoxy, a substitutedalkoxyaryloxy group, a halogen and zero;

wherein R₃ is selected from the group consisting of H, a lower alkyl andzero, and R₃ is zero when A is zero;

wherein R₁₂ is selected from the group consisting of hydrogen andmethyl;

but R₂ is not 2,5-dimethoxyphenyl when X is NH₂, Y is NH₂, Q₁ is CH, Q₂is N, Z₁ is H, B is CH, R₁ is H and R₁₂ is methyl; and

wherein each lower alkyl group is independently selected from the groupconsisting of lower alkyl groups having from about 1 to 6 carbons.

In one embodiment of formula 11, X and Y are both NH₂, Q₁ is nitrogen,Q₂ is CH, Z₁ is hydrogen, A is zero, B is nitrogen, R₁ is hydrogen, R₂is selected from the group consisting of phenyl, 2-methoxyphenyl,4-methoxyphenyl, 4-chlorophenyl, 3,4-dimethoxyphenyl,2,5-dimethoxyphenyl, 3,4,5-trimethoxyphenyl and 3,4-C₄ H₄ phenyl, R₃ iszero and R₁₂ is hydrogen.

In another embodiment of formula 11, X and Y are both NH₂, Q₁ isnitrogen, Q₂ is CH, Z₁ is hydrogen, A is zero, B is sulfur, R₁ is zero,R₂ is selected from the group consisting of 2-methoxyphenyl,4-methoxyphenyl and 3,4-dimethoxyphenyl, R₃ is zero and R₁₂ is hydrogen.

In another embodiment of formula 11, X and Y are both NH₂, Q₁ isnitrogen, Q₂ is CH, Z₁ is hydrogen, A is zero, B is nitrogen, R₁ ismethyl, R₂ is selected from the group consisting of3,4,5-trimethoxyphenyl, 2,5-dimethoxyphenyl, 3,4-dimethoxyphenyl,3,4,5-trimethoxyphenyl, phenyl, 3,4-C₄ H₄ phenyl and 2,3-C₄ H₄ phenyl,R₃ is zero and R₁₂ is hydrogen.

In another embodiment of formula 11, X and Y are both NH₂, Q₁ isnitrogen, Q₂ is CH, Z₁ is hydrogen, A is zero, B is sulfone, R₁ is zero,R₂ is selected from the group consisting of 2-methoxyphenyl,4-methoxyphenyl, and 3,4-dimethoxyphenyl, R₃ is zero and R₁₂ ishydrogen.

The present invention is also directed to pyrido 2,3-d! and3,2-d!pyrimidine compounds and pharmaceutically acceptable salts havingthe formula (12): ##STR64## wherein X and Y may be the same or differentand are selected from the group consisting of OH, NH₂, H and CH₃ ;

wherein Z and Z₁ are different and are selected from the groupconsisting of R₄ and ##STR65## where Z is R₄ when Z₁ is ##STR66## and Zis ##STR67## when Z₁ is R₄ ; wherein Q₁ and Q₂ are the same or differentand are selected from the group consisting of CH and nitrogen;

wherein A is selected from the group consisting of nitrogen, CH, sulfurand zero;

wherein B is selected from the group consisting of sulfur, sulfoxide,sulfone, CH, oxygen, nitrogen and zero; but B is not sulfur, sulfoxide,sulfone, oxygen or nitrogen when A is sulfur;

wherein R₁ is selected from the group consisting of hydrogen, a nitrosogroup, an aldehyde, a lower alkyl group, a formyl group and zero, and R₁is zero when B is zero, oxygen, sulfur, sulfoxide or sulfone;

wherein R₂ is selected from the group consisting of a lower alkyl group,p-aroyl-L-glutamate, an aryl group, an alkylaryl group, a substitutedaryl group, a substituted alkylaryl group, a diaryl group, a triarylgroup, an alkyldiaryl group, an alicyclic hydrocarbon group, anaklyltriaryl group, a substituted diaryl group and a substituted triarylgroup, and each substituent of the substituted aryl group, diaryl group,and triaryl group is the same or different and is selected from thegroup consisting of a lower alkyl group, an alkoxy, a substitutedalkoxyaryloxy group, a halogen and zero;

wherein R₃ is selected from the group consisting of H, a lower alkyl andzero, and R₃ is zero when A is zero;

wherein R₁₂ is selected from the group consisting of hydrogen andmethyl; and

wherein each lower alkyl group is independently selected from the groupconsisting of lower alkyl groups having from about 1 to 6 carbons.

For all of the formulas described above, the lower alkyl groups are thesame or different and are independently selected from those lower alkylgroups having one to six carbon atoms, such as for example methyl,ethyl, propyl, butyl, pentyl, hexyl, cyclopropyl, cyclobutyl,cyclohexyl, cyclopropylmethyl or cyclobutylmethyl groups. Alkyl groupssharing one to about six carbon atoms are preferred. These lower alkylgroups are straight chain, branched chain or cyclic (alicyclichydrocarbon) arrangements. The carbon atoms of these straight chain,branched chain or cyclic arranged alkyl groups may have one or moresubstituents for the hydrogens attached to the carbon atoms.

Suitable aryl groups include for example phenyl and benzyl groups.Suitable substituted aryl groups include for example: mono-, di- andtri-substituted alkoxy phenyl groups; mono-, di- and tri-halogenatedphenyl groups; mono-, di and tri-substituted alkyl phenyl groups; mono-,di- and tri-substituted alkoxy benzyl groups and mono-, di- andtri-substituted halogenated benzyl groups.

The term "alkylaryl" refers to groups having an alkyl moiety attached toan aryl ring such as a phenyl or benzyl ring. The alkyl moiety ispreferably a lower alkyl chain having one to about seven carbon atoms.This alkyl moiety may also contain oxygen, nitrogen or sulfur atoms,such as for example methoxy groups. The aryl moiety of the alkylarylgroup is unsubstituted, mono-substituted, di-substituted ortri-substituted. If substituted, each substituent may independently beselected from the group consisting of a lower alkyl group having one toabout seven carbon atoms, an alkoxy group such as for example a methoxygroup and a halogen, such as for example fluorine, chlorine or bromine.

Other substituent groups in the formulas 1-11 as described above are asfollows: the alkylaryl group is selected from the group consisting of analkylphenyl and alkylbenzyl group; the alkyldiaryl group is selectedfrom the group consisting of alkylnaphthyl, alkylbenzothiophene,alkylindene, alkylbenzofuran, alkylindole and alkylaminoquinoline; thealkyltriaryl group is an alkylanthracyl group; the substituted aryl,diaryl and triaryl group is selected from the group consisting of amono-, di- and tri-substituted alkylphenyl and alkylbenzyl group,alkylnaphthyl, alkylbenzothiophene, alkylindole, alkylbenzofuran,alkylindene, alkylaminoquinoline, alkylanthracyl; each substitutedalkyldiaryl and alkyltriaryl group is selected from the group consistingof a mono-, di- and tri-substituted alkylnaphthyl, alkylbenzothiophene,alkylindole, alkylbenzofuran, alkylindene, alkylaminoquinoline andalkylanthracyl group; and each substituent is the same or different andis selected from the group consisting of methyl, ethyl, n-propyl,n-butyl, branched n-pentyl, branched pentyl, n-hexyl, branched hexyl,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, methoxy group,chlorine atom, bromine atom and fluorine atom.

In some embodiments of this invention, the compounds, andpharmaceutically acceptable salts thereof, having the general formula 1,2 and 4-11 wherein X and Y are each NH₂, R₁ is selected from the groupconsisting of H, CH₃ and CHO, CH₃ CHO, and zero and R and R₂ areselected from the group consisting of 2,5-dimethoxyphenyl,2,3,4-trimethoxyphenyl, 2,4,6-trimethoxyphenyl, naphthyl,4-methoxynaphthyl, anthracyl and methoxy anthracyl, florene,benzothiophene, indene, benzofuran, indole, aminoquinoline,2,3-dichlorophenyl, 2,4-dichlorophenyl, 2,5-dichlorophenyl,2,6-dichlorophenyl, 3,4-dichlorophenyl and 3,5-dichlorophenyl. R₃ is CH₃or hydrogen, R₄ is either hydrogen, methyl, ethyl, propyl and butylgroup, cyclopropyl, cyclobutyl and cyclohexyl or zero, B is selectedfrom the group consisting of nitrogen, carbon, sulfur and oxygen, A isselected from the group consisting of nitrogen, carbon and sulfur.

In other embodiments of this invention, compounds and pharmaceuticallyacceptable salts thereof, are provided having the formulas 1, 2 and 4-8wherein X and Y are each NH₂. R₁ is selected from the group consistingof H, CH₃, NO and CHO, CH₃ CHO, zero. R and R₂ are selected from thegroup consisting of 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl,2,4-dimethoxyphenyl, 3,4-dimethoxyphenyl, 3-5-dimethoxyphenyl,2-chlorophenyl, 3-chlorophenyl, 4-chlorophenyl, 2,3,4-trichlorophenyl,2,4,5-trichlorophenyl, 2,4,6-trichlorophenyl, 3,4,5-trichlorophenyl,2-bromophenyl, 3-bromophenyl, 4-bromophenyl, 2,4-dibromophenyl,2,5-dibromophenyl, 2,6-dibromophenyl, 3,4-dibromophenyl,3,5-dibromophenyl, 2,4,6-tribromophenyl, 2-fluorophenyl, 3-fluorophenyl,4-fluorophenyl, 2,3-difluorophenyl, 2,4-difluorophenyl,2,5-difluorophenyl, 2,6-difluorophenyl, 3,4-difluorophenyl,3,5-difluorophenyl, 2,3,4-trifluorophenyl, 2,4,5-trifluorophenyl,2,4,6-trifluorophenyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl,2,3-dimethylphenyl, 2,4-dimethylphenyl, 2,5-dimethylphenyl,2,6-dimethylphenyl, 3,4-dimethylphenyl, 3,5-dimethylphenyl,2,4,5-trimethylphenyl, and 2,4,6-trimethylphenyl. R₃ is CH₃ or hydrogen,and in formulas 3-5. R₄ is selected from the group consisting ofhydrogen, methyl, ethyl, propyl, and butyl group, cyclopropyl,cyclobutyl, cyclohexyl and zero. B is selected from the group consistingof nitrogen, carbon, sulfur and oxygen and A is selected from the groupconsisting of nitrogen, carbon and sulfur.

In other embodiments of this invention, compounds and pharmaceuticallyacceptable salts thereof, are provided having the formulas 1, 2 and 4-8wherein X and Y are each NH₂. R₁ is selected from the group consistingof H, CH₃, NO and CHO, CH₃ CHO, zero. R and R₂ are selected from thegroup consisting of methyl, ethyl, n-propyl, n-butyl, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, 2-methoxybenzyl,3,4-dimethoxybenzyl, 2,3-dimethoxybenzyl, 3,4-dimethoxybenzyl,2,3,4-trimethoxybenzyl, 3,4,5-trimethoxybenzyl, 2-chlorobenzyl,3,4-dichlorobenzyl, 2,3-dichlorobenzyl, 2,4-dichlorobenzyl,2,5-dichlorobenzyl, 2,6-dichlorobenzyl, 2,6-dichlorobenzyl,3,4-dichlorobenzyl, 3,5-dichlorobenzyl, 2-bromobenzyl,3,4-dibromobenzyl, 2-fluorobenzyl, 3,4-difluorobenzyl,2,4-difluorobenzyl, 2,5-difluorobenzyl, 2,6-difliorobenzyl and3,4-difluorobenzyl. R₃ is CH₃ and hydrogen, and in formulas 3-5 R₄ isselected from the group consisting of a hydrogen, methyl, ethyl, propyl,butyl group, cyclopropyl, cyclobutyl, cyclohexyl; B is selected from thegroup consisting of nitrogen, carbon, sulfur and oxygen; and A isselected from the group consisting of nitrogen, carbon and sulfur.

The present invention further relates to methods of using theabove-described compounds, and pharmaceutically acceptable saltsthereof, in therapeutically an/or prophylactically treating a patientwith an illness. As used herein, the term "illness" refers to cancer,infection by Pneumocystis carinii, infection by Toxoplasmosis gondii, orother secondary infections arising in immunocompromised patients, suchas those with AIDS, and infections caused by bacteria, malaria, fungi orprotozoa.

A method of therapeutically treating a patient for an illness comprisesthe steps of:

a) employing a compound, or pharmaceutically acceptable salts thereof,having any of the above formulas 1, 2 or 4-11;

b) incorporating said compound in a suitable pharmaceutical carrier; and

c) administering a therapeutically effective amount of said compoundincorporated in said carrier to a patient.

As used herein, the term "suitable pharmaceutical carrier" refers to anypharmaceutical carrier known in the art, absent compatibility problemswith the compounds of formula 1, 2 or 4-11. Preferred carriers includephysiologic saline and 5% dextrose.

As used herein, the term "therapeutically effective amount" refers tothat amount of any of said compounds of formulas 1, 2 or 4-11incorporated in a suitable pharmaceutical carrier which is required tobring about a desired effect, such as reducing tumor size, destroyingcancerous cells or resisting/treating infection caused by organisms suchas Pneumocystis carinji and Toxoplasmosis gondii.

As will be understood by one skilled in the art, a therapeuticallyeffective amount of said compound can be administered by any means knownin the art, including but not limited to, injection, parenterally,orally, or, where appropriate, topically.

It is well within the skill of one practicing in the art to determinewhat dosage, and the frequency of this dosage, which will constitute atherapeutically effective amount for each individual patient, dependingon the type of illness and the severity of such illness. It is alsowithin the skill of one practicing in the art to select the mostappropriate method of administering the compounds based upon the needsof each patient.

Methods of prophylactically treating a patient for an illness comprisethe steps of:

a) employing a compound, or pharmaceutically acceptable salts thereof,having any of the formulas 1, 2 or 4-11 as described above;

b) incorporating said compound in a suitable pharmaceutical carrier; and

c) administering a prophylactically effective amount of said compoundincorporated in said carrier to a patient; wherein said illness isselected from the group consisting of infection caused by Pneumocystiscarinii and Toxoplasmosis gondii.

As used herein, the term "prophylactically effective amount" refers tothat amount of any of the compounds described above which will cause thebody to generate antibodies in amounts sufficient to resist the onset ofinfection caused by Pneumocystis carinii or Toxoplasmosis gondii inimmunocompromised patients.

As will be understood by one skilled in the art, a prophylacticallyeffective amount of said compound can be administered by any means knownin the art, including but not limited to, injection, parenterally,orally, or, where appropriate, topically.

It is well within the skill of one practicing in the art, to determinewhat dosage, and the frequency of this dosage, which will constitute aprophylactically effective amount for each individual patient, dependingon the type of illness, such as the type of cancer, and the severity ofsuch illness. It is also within the skill of one practicing in the artto select the most appropriate method of administering the compoundsbased upon the needs of each patient.

EXAMPLES

The following examples are set forth to illustrate various embodimentsof the invention, and should not be construed as limiting the inventionin any way. Standard test procedures familiar to those skilled in theart were used in the examples, such as those procedures described byGangjee, A., et al., in "5-Arylthio-substituted 2-amino-4-oxo-6-methylpyrrolo!2,3-d! pyrimidine antifolates as thymidylate synthase inhibitorsand antitumor agents", J. Med. Chem., Vol. 38, pp. 4495-4502 (1995);"Effect of bridge region variation on antifolate and antitumor activityof classical 5-substituted 2,4-diamino furo 2,3-d! pyrimidines", J. Med.Chem., Vol. 38, pp. 3798-3805 (1995); and "Novel2,4-diamino-5-substituted-pyrrolo 2,3-d!pyrimidines As Classical andNon-Classical Antifolate Inhibitors of Dihydrofolate Reductases", J.Med. Chem., Vol. 38, pp. 2158-2165 (Jun. 6, 1995) and referencesdisclosed therein.

Example 1

Compounds 1-6, 8, 301 and 303-312 were evaluated as inhibitors ofdihydrofolate reductase (DHFR) from Pneumocystis carinii (Pc),Toxoplasmosis gondii (Tg) and rat liver (RL). Performance of thesecompounds was compared with that of trimetrexate (TMQ), piritrexim(PTX), trimethoprim (TMP) and methotroxate (MTX), all of which arecurrently available. Trimetrexate is available from Warner-Lambert/ParkeDavis Pharmaceutical Research, Ann Arbor, Mich. Trimetrexate is approvedby the United States Food and Drug Administration as an approved newdrug for the treatment of Pneumocystis carinii infections in patientswith AIDS. PTX is an experimental anticancer agent in Phase II clinicaltrials and is also an agent against Pneumocystis carinii andToxoplasmosis gondii. TMP is an agent used against Pneumocystis cariniiinfection in conjunction with sulfonamides. MTX is a clinical usedanticancer agent.

The evaluations of Compounds 1-6, 8, 301 and 303-312 consisted ofdetermining the IC₅₀ values and selectivity ratios of each compoundagainst Pc DHFR, Tg DHFR and RL DHFR. The IC₅₀ value is theconcentration of a compound required to inhibit the dihydrofolatereductase activity by 50 percent (%). It will be understood by thoseskilled in the art that the lower the IC₅₀ value the more potent thecompound. The selectivity ratio is a measure of the selectivity of acompound for Pc DHFR or Tg DHFR and is expressed as the IC₅₀ value ofthe DHFR from rate liver (RL) divided by the IC₅₀ value of either the PcDHFR or the Tg DHFR, depending on which organism the compounds are beingtested against. For example, the selectivity ratio of a compound iscalculated by the following formula: ##EQU1##

It will be understood by those skilled in the art that the higher theselectivity ratio, the less toxic the compound is to mammaliandihydrofolate reductase, and thus, less toxic to the patient.

Table 4 sets forth the IC₅₀ values for Pc DHFR, RL DHFR and Tg DHFR andthe corresponding selectivity ratios for the compounds tested.

                  TABLE 4    ______________________________________    Inhibitory Concentrations (IC.sub.50 μM) and Selectivity Ratios                           Selectivity                           Ratio:         Selectivity    Com-           RL      RL DHFR/Pc                                    Tg    Ratio:    pound #          Pc DHFR.sup.1                   DHFR.sup.1                           DHFR     DHFR.sup.1                                          RL DHFR/Tg    ______________________________________     1    >23      56.3    <2.4     8.1   7.0     2    119.0    116.0   1.0      4.3   27.0     3    279.0    63.0    0.23     6.0   10.5     4    45.7     156.0   3.4      1.7   92.0     5    >21      70.0    <3.3     5.3   13.2     6    35.3     14.4    0.4      1.4   10.3     8    252.0    >252    >1       3.9   >65     9    0.038    0.044   1.20     0.21  0.21    301   307.0    59.3    0.2      1.1   53.9    303   81.0     4.2     0.05     1.4   3.0    304   >12.0    >12.0   ND*      3.4   >4.0    305   28.90    3.0     0.11     1.0   3.0    306   209.0    8.20    0.04     0.87  9.43    307   11.10    16.7    1.50     2.60  6.42    308   58.50    5.30    0.09     11.6  0.46    309   10.60    3.00    0.28     0.81  3.70    310   929.0    82.9    0.09     9.20  9.01    312   0.044    0.06    1.36     0.15  0.40    TMQ   0.042    0.003   0.07     0.01  0.30    PTX   0.038    0.001   0.04     0.01  0.14    TMP   12.0     133.0   11.1     2.7   49.0    MTX   0.001    0.003   3.0      0.014 0.21    ______________________________________     *ND = not determined

As can be seen from the above table, Compound 4 is almost two-times asselective at TMP and 306 times as selective as TMQ. Compound 8 similarlyshowed much higher selectivity than the compounds known in the art.These two compounds, therefore, represent preferred embodiments of theinvention for the treatment of infections caused by Pneumocystis cariniiand Toxoplasmosis gondii as well as in treatment of cancer patients asan antitumor agent or to destroy cancerous cells. With regard toPneumocystis carinii, Compounds 4 and 8, with their high potency andhigh selectivity, may be used clinically with a lesser amount of aleucovorin as compared to TMQ or may be used clinically without thenecessity of leucovorin, and thus greatly reduce the cost ofadministering these compounds to a patient.

Example 2

Compound 9 was tested for DHFR inhibition against the growth of humanleukemia CCRF-CEM cells. The performance of Compound 9 was evaluatedagainst MTX. Four different types of cells were used: CCRF-CEM cellswere unaltered; R30dm cells had a decreased amount of polyglutamatesynthase; R1 cells had a twenty-fold increase in wild type DHFR proteinand activity; and R2 cells were deficient in their ability to uptake thecompounds into the cell. Results are presented in Table 5 below.

                  TABLE 5    ______________________________________    Growth Inhibition of Parental CCRF-CEM and Sublines with    Single, Defined Mechanisms of MTX Resistance During Continuous    (0-120 hours) Exposure to MTX and Compound 311 (EC.sub.50 in nM)    Compound            CCRF-CEM   R30dm     R1      R2    ______________________________________    MTX     14.5 ± 0.4                       14.5 ± 0.5                                 595 ± 5                                         3100 ± 100            (n = 5)    (n = 2)   (n = 2) (n = 2)    311     12.8 ± 2.2                       36 ± 1 515 ± 25                                         1650 ± 200            (n = 5)    (n = 2)   (n = 2) (n = 2)    ______________________________________     Average values are presented ± the standard deviation range for n = 2     and ± the standard deviation range for n ≧ 3.

As can be seen from the results of the above table, Compound 9 performedcomparably to MTX. The example further demonstrates thatpolyglutamylation may play a limited role in the mechanism of action ofCompound 9, even in continuous exposure. Both the R1 subline, withamplified DHFR expression, and the MTX-transport deficient R2 sublinedisplayed resistance to MTX under continuous exposure.

Example 3

Compound 9 and MTX were also tested for growth inhibition of A253 andFaDu human squamous carcinoma cell lines; neither of these cell lineshad any deficiencies. Results are presented below in Table 6.

                  TABLE 6    ______________________________________    Growth Inhibition of A253 and FaDu Human Squamous    Carcinoma Cell Lines Following Continuous (120 hours) Exposure    to MTX and Compound 311 (EC.sub.50 in nM)    Compound        A253    FaDu    ______________________________________    MTX             17 ± 1                            31 ± 2    311             46 ± 4                            22 ± 4    ______________________________________     All values are average ± standard deviation range for duplicate     determinations.

Example 4

Compound 9 and aminopterin were further tested for their activity assubstrates for human leukemia cell CCRF-CEM folylpolyglutamatesynthetase. Aminopterin is a classical 2,4-diamino antifolate substrate.Results are presented below in Table 7.

                  TABLE 7    ______________________________________    Activity of Aminopterin and Compound 9 as Substrates for    CCRF-CEM Human Leukemia Cell Folylpolyglutamate synthetase    Substrate KM, μM                       V.sub.max, rel                                   V.sub.max /K.sub.m(rel)                                           n    ______________________________________    Aminopterin              4.3 ± 0.2                       1           0.23 ± 0.01                                           3    9         <1       0.72 ± 0.07                                   >0.72   4    ______________________________________     Values presented are average ± standard deviation.

This examples demonstrates that Compound 9 is a potent tumor cell growthinhibitor that shares determinates of response with MTX. It is similarin potency to MTX as an inhibitor of the growth of human leukemia andSCC cell lines in culture. DHFR is suggested as the target of Compound9, which is supported by the data showing relatively potent DHFRinhibition in vitro and the cross-resistance to Compound 9 of a humancell line having amplified expression of DHFR. Compound 9 may also usethe MTX/reduced folate-transport protein for uptake as evidenced by thecross-resistance of a human cell line in which this transport system isdefective. This example indicates that Compound 9 may be an excellentsubstrate for human FPGS with a very low K_(m). The slightcross-resistance to continuous exposure of the cell line having lowlevels of FPGS suggests that polyglutamate metabolites may play a rolein growth inhibition even under these conditions.

Example 5

Compound 9 was further tested for its in vitro anti-cancer activity bythe National Cancer Institute. Results are presented in Table 8 below.GI₅₀ represents the concentration at which growth was inhibited by 50%.

                  TABLE 8    ______________________________________    In Vitro Anti-Cancer Activity of Compound 311    Panel/Cell Line   GI.sub.50    ______________________________________    Leukemia    CCRF-CEM          ND    HL-60 (TB)        ND    MOLT-4            ND    RPMI-8226         ND    SR                >1.00E-04    Non-Small Cell Lung Cancer    A549/ATCC         <1.00E-08    EKVX               3.97E-05    HOP-62            <1.00E-08    HOP-92             8.40E-06    NCI-H226          >1.00E-04    NCI-H23            5.75E-08    NCI-H322M         >1.00E-04    NCI-H522          >1.00E-04    Colon Cancer    COLO 205          >1.00E-04    HCC-2998          <1.00E-08    HCT-116           <1.00E-08    HCT-15            <1.00E-08    HT29              <1.00E-08    KM12               3.32E-05    SW-620             3.15E-06    CNS Cancer    SF-268            <1.00E-08    SF-295            <1.00E-08    SF-539            <1.00E-08    SNB-19            >1.00E-04    SNB-75            <1.00E-08    U251              <1.00E-08    Melanoma    LOX IMVI          <1.00E-08    MALME-3M          <1.00E-08    M14               <1.00E-08    SK-MEL-2          >1.00E-04    SK-MEL-28         >1.00E-04    UACC-257          ND    UACC-62           <1.00E-08    Ovarian Cancer    IGROV1            <1.00E-08    OVCAR-3           >1.00E-04    OVCAR-4           >1.00E-04    OVCAR-5           >1.00E-04    OVCAR-8           <1.00E-08    SK-OV-3           >1.00E-04    Renal Cancer    786-0             <1.00E-08    ACHN              <1.00E-08    CAKI-1            <1.00E-08    RXF 393           >1.00E-04    SN12C             <1.00E-08    TK-10             >1.00E-04    UO-31             <1.00E-08    Prostate Cancer    PC-3              >1.00E-04    DU-145             1.09E-08    Breast Cancer    MCF7              <1.00E-08    MCF7/ADR-RES      <1.00E-08    MDA-MB-231/ATCC   >1.00E-04    HS 578T           >1.00E-04    MDA-MB-435         6.87E-07    MDA-N             <1.00E-08    BT-549            >1.00E-04    T-47D             >1.00E-04    ______________________________________

As will be appreciated by one skilled in the art, the lower the GI₅₀,the more effective the compound. A GI₅₀ of less than 1.00×10⁻⁸ indicatesa very high potency, whereas a GI₅₀ greater than 1.00×10⁻⁴ indicatesthat the compound was relatively inactive.

As demonstrated in Table 8 above, Compound 9 was shown to be highlyselective against certain cell lines, with little or no activity againstother cell lines. This result indicates that Compound 9 is not a generalpoison, but rather is very selective. For example, in the eight breastcancer cell lines, Compound 9 was found to be highly potent, with GI₅₀of less than 1.00×10⁻⁸ M, against the MCF7, MCF7-ADR-RES (resistant cellline) and the MDA-N cell lines, but was relatively inactive, with GI₅₀greater than 1.00×10⁻⁴ M against breast cancer cell linesMDA-MB-231/ATCC, HS578T, BT-549 and T-47D. The difference in activity ofthese two sets of breast cancer cells is ten thousand fold, whichindicates a very high degree of selectivity. This kind of selectivity isrepeated in all of the cell lines tested.

Example 6

The inhibitory concentration of Compounds 313 and 314 against TS wasdetermined. The performance of these compounds was measured againstPDDF, which is a standard TS inhibitor and experimental anticanceragent. The compounds were all tested against Lactobacillus casei andhuman lymphoma cells. Results are presented in Table 9 below:

                  TABLE 9    ______________________________________    Inhibitory Concentrations (IC.sub.50 in μM) Against TS    Compound       L. Casei TS                             Human TS    ______________________________________    313            180       180    314            360       ND    PDDF           0.036     0.036    ______________________________________

As can be seen from the above table, Compound 313 and 314, when tested,had surprisingly high inhibitory concentration when compared with thatof PDDF.

Example 7

Compounds 315 and 317-322 were tested for their inhibitory activityagainst TS. The performance of these compounds was measured againstZD1694, which is in Phase III clinical trials as an antitumor agent. Allof the compounds were tested against human cells, L. casei, E. coli, andS. faecium. Results are presented in the Table 10 below.

                  TABLE 10    ______________________________________    Inhibitory Concentrations (IC.sub.50 in μM) Against TS    Compound Human    L. Casei  E. Coli S. Faecium    ______________________________________    ZD1694   0.22     8.8       5.3     8.8    315      >25      >26       ND      ND    317      2.4      >24 (33%) >24 (30%)                                        >24 (12%)    318      0.13     45        45      >45 (31%)    319      1.0      >26 (0%)  >26 (40%)                                        30    320      0.15     5.1       13      15    321      30       >30 (0%)  >30 (30%)                                        >30 (20%)    322      2.0      >25 (32%) >25 (36%)                                        >25 (0%)    ______________________________________

Example 8

Compounds 317-320 were also tested for their ability to inhibit DHFR.Selectivity ratios were determined as measured against Pneumocystiscarinii (Pc), Toxoplasmosis gondii (Tg) and rat liver (RL). Results arepresented in Table 11 below.

                  TABLE 11    ______________________________________    Inhibition of Dihydrofolate Reductase (IC.sub.50 in μM)                             Selectivity                             Ratio        Selectivity    Compound Pc      RL      RL/Pc   Tg   Ratio RL/Tg    ______________________________________    317      >21     >21     ND       20  >1    318      >20     >20     ND      11.7 >1.7    319       40     24.6    0.62     3.1 7.94    320      >15     7470    ND      244  30.61    ______________________________________

As can be seen from Table 11, Compound 320 is highly selective forToxoplasmosis gondii DHFR.

Example 9

Compounds 317-322 were also tested for their ability to inhibit thegrowth of FaDu human squamous cell carcinoma cell lines. The performanceof these compounds was tested against PDDF, and AG331, which is a TSinhibitor in Phase III clinical trials as an antitumor agent. Resultsare presented in Table 12 below.

                  TABLE 12    ______________________________________    Growth Inhibition of the FaDu Human Squamous Cell Carcinoma    Cell Line By Continuous (120 hours) Exposure to the Inhibitors    Compound         EC.sub.50 μM                              n    ______________________________________    317              >10      2    318              insoluble                              --    319              6.7 ± 1.5                              3    320              1.5 ± 0.4                              3    321              >10      2    322              ND       --    PDDF             1.7 ± 0.2                              4    AG331            1.0 ± 0.1                              6    ______________________________________     Values presented are average ± standard deviation.

Example 10

The following example describes methods of synthesizing the compoundsrepresented by formula 2. These methods are illustrated in FIG. 1.Reference numerals and letters correspond with those of FIGS. 1 and 2.

2-amino-3,4-dicyanopyrrole (11)

A mixture of about 4.0 grams (g) of Compound 10, about 4.0 g of 5% Pd onBaCO₃, about 15 milliliters (ml) of DMF and about 25 ml methanol washydrogenated at about 50 psi for approximately 3 hours. The mixture wasfiltered through Celite®, a filtering composition commercially availablefrom Johns-Mannville Products Corporation, and the filtrate concentratedunder reduced pressure to about 10 ml. About 200 ml of cold water wasadded to the concentrate, and a light brown solid was formed. This solidwas collected by filtration to yield about 1.60 g of Compound 11.

2,4-diamino-5-cyanopyrrolo 2,3-d!pyrimidine (12)

A mixture of about 2.63 g of Compound 11 and about 2.5 g ofchlorformamidine hydrochloride in about 50 ml Dowtherm-A®, a liquid heattransfer media commercial available from Dow Chemical Company, washeated at between about 160° and 170° C. for approximately 48 hours,until Compound 11 could not be detected by thin layer chromatography(TLC). The mixture was cooled to room temperature, and about 50 ml ofEt₂ O was added thereto. A greenish-brown solid resulted. The solid wasfiltered and washed with Et₂ O to yield about 3.0 g of Compound 12.

2,4-diaminopyrrolo 2,3-d!pyrimidine-5-carboxaldehyde (13)

About 6.0 g of Raney Ni was added to a stirred solution of about 2.0 gof Compound 12 and about 50 ml of HCOOH. The mixture was heated to about80° C. for about 2 hours, until no starting material could be detectedby TLC. The mixture was cooled to room temperature and filtered throughCelite®. The filtrate was evaporated under reduced pressure, azeotropingwith methanol to remove traces of HCOOH. The residue was dissolved inabout 25 ml of hot water, treated with Norit®, an activated adsorptioncarbon commercially available from American Norit Company, Inc., andfiltered through Celite®. The filtrate was neutralized with NH₄ OH. Thelight brown precipitate which resulted was filtered and dried to yieldabout 1.40 g of Compound 13, which was immediately used in subsequentreactions without further purification.

2,4-diamino-5N-(3',4',5'-trimethoxyphenyl)imino!methyl!pyrrolo!2,3-d!pyrimidine (14a)

A solution containing about 1.30 g of Compound 12 and about 2.06 g of3,4,5-trimethoxyaniline in about 75 ml of 70% acetic acid and containingabout 6.50 g of damp Raney Ni was hydrogenated at atmospheric pressurefor about 24 hours at room temperature. The mixture was treated withNorit® and filtered through Celite® and the solvent removed from thefiltrate by evaporation under reduced pressure. About 15 ml of coldwater was added to the residue, and the suspension was added to about100 ml of a stirred, cold, saturated solution of NaHCO₃. The mixture wasstirred for about 10 minutes and refrigerated for about 6 hours. Thebrown precipitate which formed was collected, washed with water, anddried. This product, containing Compound 14A, was washed repeatedly withEt₂ O until no aniline was detected by TLC in the washings. The residuewas then dissolved in about 100 ml methanol and filtered, and thefiltrate evaporated under reduced pressure to near dryness. About 50 mlEt₂ O was added to the solution, and the precipitate filtered to yieldabout 1.20 g of Compound 15a.

2,4-diamino-5- (3',4',5'-trimethoxyanilino)methyl!pyrrolo2,3-d!pyrimidine (1)

About 0.05 g NaCNBH₃ were added to a solution containing about 0.20 g ofCompound 15a in about 25 ml methanol. The pH was adjusted to about 2with a 50% methanol/hydrochloric acid solution. The mixture wascontinuously stirred at room temperature for about 4 hours. The solventwas evaporated to dryness, and cold water was added to the residue,which was neutralized with NH₄ OH. The resulting precipitate wasfiltered, dried, and dissolved in a 9:1 mixture of CHCl₃ /methanol. Thiswas applied to a silica gel column (2.4 cm×20 cm) packed in CHCl₃. Thecolumn was eluted with a gradient of 1% methanol in CHCl₃ to 15%methanol in CHCl₃. Fractions corresponding to the product, as determinedby TLC, were pooled and evaporated to dryness under reduced pressure.The residue was triturated in cold Et₂ O and filtered to yield about0.10 g of Compound 1.

2,4-diamino-5- (3',4'-dimethoxyanilino)methyl!pyrrolo 2,3-d!pyrimidine(2)

The Schiff base was prepared as described for Compound 15a, except thatthe reaction was carried out in 80% acetic acid and using3,4-dimethoxyaniline to yield about 1.20 g of Compound 15b. Reduction ofCompound 15b was carried out as described for Compound 1. The crudeproduct was dissolved in methanol and filtered. The filtrate wasevaporated under reduced pressure to dryness. The residue was trituratedin cold Et₂ O and filtered to yield about 0.51 g of Compound 2.

2,4-diamino-5- (4'-methoxyanilino)methyl!pyrrolo 2,3-d!pyrimidine (3)

The Schiff base was prepared as described above for Compound 15b using4-methoxyaniline to yield about 0.32 g of Compound 15c. Reduction ofCompound 15c was carried out as described for Compound 15b to yieldCompound 3.

2,4-diamino-5- (2',5'-dimethoxyanilino)methyl!pyrrolo 2,3-d!pyrimidine(4)

The Schiff base was prepared as described above for Compound 15b using2,5-dimethoxyaniline to yield about 0.90 g of Compound 15d. Reduction ofCompound 15d was carried out as described above for Compound 15b toyield about 0.25 g of Compound 4.

2,4-diamino-5- (2',5'-diethoxyphenyl)imino!methyl!-pyrrolo2,3-d!pyrimidine(15e)

Method A, starting from Compound 13: A solution of about 1.15 g ofCompound 13 and about 1.76 g of 2,5-diethoxyaniline (14e) in about 75 ml70% acetic acid containing about 5.75 g of damp Raney Ni washydrogenated at about 55 psi for about 12 hours at room temperature. Themixture was filtered through Celite® and the filtrate evaporated underreduced pressure. About 15 ml cold water were added to the residue andthis suspension was added to about 100 ml of a stirred, cold, saturatedsolution of NaHCO₃. The mixture was stirred for an additional 10 minutesand refrigerated for about 4 hours. The brown precipitate which resultedwas collected, washed with water, and dried. The crude productcontaining Compound 14e was washed repeatedly with Et₂ O until noaniline could be detected by TLC in the washings. The residue was thendissolved in about 100 ml methanol and filtered, and the filtrateevaporated to dryness under reduced pressure. About 20 ml Et₂ O wasadded to the solution, and the precipitate filtered to yield about 1.20g of Compound 15e.

Method B, starting from Compound 12: The Schiff base was prepared asdescribed above for Compound 15b using 2,5-diethoxyaniline to yieldabout 0.90 g of Compound 15e, which was identical in all respects withthe sample prepared according to Method A described above.

2,4-diamino-5- (2',5'-diethoxyanilino)methyl!pyrrolo 2,3-d!pyrimidine(5)

Reduction of Compound 15e was performed as described above for Compound15b to yield about 0.36 g of Compound 5.

2,4-diamino-5- (3',4'-dichloroanilino)methyl!pyrrolo 2,3-d!pyrimidine(6)

The Schiff base was prepared as described above for Compound 15b using3,4-dichloroaniline to yield about 1.0 g of Compound 15f. Reduction ofCompound 15f was performed as described above for Compound 15b to yieldabout 0.34 g of Compound 6.

2,4-diamino-5- (1'-naphthylamino)methyl!pyrrolo 2,3-d!pyrimidine(7)

The Schiff base was prepared as described above for Compound 15b, exceptthat the reaction was carried out at about 30 psi for about 12 hours atroom temperature using 1-aminonaphthylene to yield about 0.92 g ofCompound 15 g. Reduction of Compound 15 g was carried out as describedabove for Compound 15b, except that glacial acetic acid was used toadjust the pH to about 2. Crude product 7 was dissolved in a 9:1 mixtureof CHCl₃ /methanol, which was loaded on a silica gel column (2.4 cm×20cm) packed in CHCl₃. The column was eluted with a gradient of 1%methanol in CHCl₃ to 5% methanol in CHCl₃. Fractions corresponding tothe product, as determined by TLC, were pooled and evaporated underreduced pressure to dryness. The residue was triturated in cold Et₂ Oand the suspension filtered to yield about 0.24 g of Compound 7.

2,4-diamino-5-(anilinomethyl)pyrrolo 2,3-d!pyrimidine(8)

The Schiff base was prepared as described above for Compound 15b usinganiline to yield about 0.69 g of Compound 15h. Reduction of Compound 15hwas carried out as described for Compound 15b, except that glacialacetic acid was used to adjust the pH to about 2. About 0.19 g ofCompound 8 resulted.

N- 4- N-!(2,4-diaminopyrrolo2,3-d!pyrimidin-5-yl)methyl!amino!benzoyl!-L-glutamic acid (9)

The Schiff base was prepared as described above for Compound 15b usingdiethyl(p-aminobenzoyl)-L-glutamate to yield about 1.39 g of thediethylester of Compound 16. Reduction of Compound 16 was carried out asdescribed above for Compound 15b to yield about 0.44 g of Compound 17.Hydrolysis of the esters was carried out by stirring a solution of about0.30 g Compound 17 in about 10 ml of IN sodium hydroxide and 10 mlmethanol for about 72 hours at room temperature. The solvent wasevaporated to 5 ml, and the mixture was carefully acidified with glacialacetic acid in an ice bath. The tan precipitate which resulted wasfiltered, washed with water, and dried to yield about 0.23 g of Compound9.

Example 11

The DHFR inhibitory concentrations of Compounds 159-169, 171, 172, 175,177, 178, and 179 was determined. Performance of these compounds wasmeasured against TMQ and TMP. Inhibitory concentration was measuredagainst Pneumocystis carinii and Toxoplasmosis gondii, as well as ratliver. Results are presented in Table 13 below.

                  TABLE 13    ______________________________________    Inhibitory Concentrations (IC.sub.50, μM) and    Selectivity Ratios of 5-substituted furo 2,3-d!pyrimidines                                            Selectivity    Com-                     Selectivity    Ratio    pound #           Pc DHFR  RL DHFR  Ratio RL/Pc                                     Tg DHFR                                            RL/Tg    ______________________________________    159    >26      252      ND      >26    ND    160    19       23       1.2     19     1.2    161    0.65     12.3     18.9    11.6   1.1    162    13.5     12       0.89    37     0.32    163    41       36.5     0.89    38     0.96    164    14       60.3     4.31    >42    ND    165    >12      >12      ND      >12    ND    166    8.1      16.2     2.00    32.4   0.50    167    7.7      187      17.79   45.4   3.02    169    50.9     71.9     1.4     >47    ND    171    44.8     >27      ND      >27    ND    172    284      34.3     0.1     21.5   1.6    175    >31.3    >31.3    ND      >31.3  ND    177    8.6      >83      >10     >83    ND    178    >12      >12      ND      >12    ND    179    >27.9    >27.9    ND      >27.9  ND    TMQ    0.042    0.003    0.07    0.01   0.30    TMP    12       133      11.1    2.7    49.0    ______________________________________

As can be seen from the above table, Compound 161 is approximately 18times more active than TMP and 1.7 times more selective; Compound 161 is271 times more selective than TMQ and only 15 times less potent. Thesecompounds, therefore, exhibit high selectivity for Pc DHFR. Compound 167was also more selective than the compounds known in the art.

Example 12

The following example provides methods for synthesizing compounds offormula 4. Reference numerals correspond with those in FIGS. 6 and 7.

Methyl 4- 2-(2,4-diaminofuro 2,3-d!pyrimidin-5-yl)-ethanyl!benzoate(27)

About 2.64 g of tributylphosphine was added to a solution containingabout 0.79 g of 2,4-diamino-5(chloromethyl)furo 2,3-d!pyrimidine (25) inabout 10 ml anhydrous DMSO. The mixture was stirred at about 60° C. forabout 2 hours under nitrogen to form the phosphonium salt. The solutionwas cooled to room temperature, at which time 0.35 g of sodium hydride(60% dispersion in mineral oil) followed by about 0.72 g of methyl4-formylbenzoate (26) was added. The mixture was stirred for about 24hours at room temperature. The DMSO was removed by vacuum distillation.About 50 ml of ethylether was added to the residue and the supernatantdecanted after 10 minutes. About 50 ml of ethylether was added again,the residue stirred for about 1 hour, and the supernatant decanted. Thisprocess was repeated 3 more times; the mixture was then stored at 0° C.with about 50 ml ethylether. After about 18 hours, the mixture wasultrasonicated for 2 hours and cooled to 0° C. for a period of about 10hours. The resulting solid was filtered, washed with ethylether, airdried, washed with water and air dried again. The solid was thensuspended in about 250 ml of hot methanol. About 3 g of silica gel wereadded to the filtrate, and the suspension was evaporated to drynessunder reduced pressure. The silica gel plug was loaded on a dry silicagel column (2.4×20 cm) and flushed initially with CHCl₃ (500 ml). Thecolumn was then eluted sequentially with 100 ml portions of 1% to 8%methanol in CHCl₃. Fractions which showed a major spot at R_(f) 0.66, asdetermined by TLC, were pooled and evaporated to dryness. The resultingresidue was dissolved in glacial acetic acid and evaporated to dryness.This residue was redissolved in hot methanol and the solution stored at0° C. for about 72 hours. The resulting solid was filtered, washed withether, and dried to yield about 0.52 g of Compound 27.

Methyl 4- 2-(2,4-diaminofuro 2,3-d!pyrimidin-5-yl)ethyl!benzoate (28)and (+)-methyl 4-!2-(2,4-diamino-5,6-dihydrofuro2,3-d!pyrimidin-5-yl)ethyl!benzoate(29)

About 0.31 g of 5% palladium on carbon was added to a solutioncontaining about 0.155 g of Compound 27 in about 20 ml of a 1:1 mixtureof methanol/DMF. The suspension was hydrogenated in a Parr apparatus atroom temperature and 25 psi of hydrogen pressure for 30 minutes. Thereaction mixture was filtered through Celite®, and the catalyst waswashed with about 30 ml of a 1:1 methanol/DMF mixture. The filtrate wasevaporated to dryness under reduced pressure, and the residue dissolvedin about 100 ml methanol. About 500 mg of silica gel was added to thesolution, and evaporated to dryness. The silica gel plug was loaded on adry silica gel column (2.4×16 cm) and flushed with about 500 ml CHCl₃.The column was then eluted with a gradient of 1-9% methanol in CHCl₃,collecting 15 ml fractions. Fractions showing a single spot at R_(f)0.63, as determined by TLC, were pooled and evaporated to dryness, andthe residue was stirred in ether, filtered, and dried to yield about0.08 g of Compound 28. Later fractions from the column described above,showing a single spot at R_(f) 0.52, were pooled and evaporated todryness under reduced pressure, and the residue obtained was stirred inether, filtered, and dried to yield about 0.02 g of Compound 29.

4- 2-(2,4-diaminofuro 2,3-d!pyrimidin-5-yl)ethyl!benzoic acid (30)

About 1.5 ml of 1N sodium hydroxide was added to a solution containingabout 0.065 g of Compound 28 in about 10 ml of a mixture of 2:1methanol/DMSO. The mixture was stirred at room temperature for 18 hoursand evaporated to dryness under reduced pressure (oil pump). The residuewas dissolved in about 5 ml water and 1N HCl was added dropwise to bringthe pH of the solution to 5.5. The suspension was cooled to 5° C. forabout 12 hours and filtered. The residue was washed sequentially withwater, acetone and ether and dried to yield about 0.05 g of Compound 30.

N- 4- 2-(2,4-diaminofuro2,3-d!pyrimidin-5-yl)ethyl!-benzoyl!-L-glutamicacid (33)

About 45 microliters of triethylamine was added to a suspensioncontaining about 0.047 g of Compound 30 in about 3 ml anhydrous DMF, andthe mixture stirred under nitrogen at room temperature for about 5minutes. The solution was cooled to 0° C., about 42 microliters ofisobutylchloroformate was added, and the mixture stirred at 0° C. for 30minutes. About 0.077 g of diethyl-L-glutamate hydrochloride was added tothe reaction mixture, followed immediately by about 45 microliterstriethylamine. The mixture was slowly allowed to warm to roomtemperature, and stirred under nitrogen for a period of about 18 hours.The reaction mixture was then subjected to another cycle of activationusing 1/2 of the quantities listed above. The reaction mixture waswarmed to room temperature and stirred for 24 hours and evaporated todryness under reduced pressure. The residue was dissolved in a 4:1mixture of CHCl₃ /methanol and chromatographed on a silica gel column(2.4×15 cm), packed with CHCl₃ /methanol (24:1), eluting with 24:1 CHCl₃/methanol. Fractions showing a single spot were pooled and evaporated todryness. The residue was stirred in cold anhydrous ether and filtered toobtain about 0.054 g of Compound 31. About 1 ml of 1N sodium hydroxidewas added to a solution containing about 0.052 g of Compound 31 in about5 ml methanol and the solution stirred at room temperature for 24 hours.The methanol was evaporated under reduced pressure, the residuedissolved in about 5 ml water, and stirring was continued for anadditional 24 hours. The pH of the solution was then adjusted to 4.0 bydropwise addition of 1N HCl. The resulting suspension was stored at 5°C. for about 12 hours and filtered; the residue was washed well withwater and acetone and dried to yield about 0.044 g of Compound 33.

4- N-(tert-butyloxycarbonyl)amino!methyl!benzoic acid (39)

About 10 ml of 1N sodium hydroxide was added to a solution containingabout 1.51 g of 4-(aminomethyl)benzoic acid (32) in about 20 ml of 1:1dioxane/water. The mixture was stirred at room temperature for about 12hours, and evaporated to half of its original volume under reducedpressure. The pH of the solution was adjusted to 3 by dropwise additionof 50% aqueous HCl, while maintaining the temperature below 10° C. withan ice bath. The resulting suspension was diluted with water (70 ml) andextracted with ethylacetate (3×50 ml). The combined organic layers werewashed with about 50 ml saturated NaCl, dried MgSO₄, and filtered. Thefiltrate was evaporated to dryness under reduced pressure and theresidue recrystallized from a mixture of ethylacetate/hexanes to yieldabout 2.10 g of Compound 39.

Diethyl-N- 4- N-(tert-butyloxycarbonyl)amino!methyl!benzoyl!-L-glutamate(34)

A solution containing about 1.26 g of Compound 39 in about 20 mlanhydrous DMF under nitrogen was cooled in an ice-salt bath. About 0.55ml of N-methylmorpholin was added to the cooled solution, followed 5minutes later by about 0.65 ml isobutylchloroformate. After stirring fora period of about 20 minutes, 1.20 g diethyl-L-glutamate hydrochloridewas added, followed immediately by about 0.55 ml N-methylmorpholin. Thereaction mixture was warmed to room temperature and stirred for 12hours. At this time, the activation cycle was repeated using 1/2 theamounts of reagents indicated above, after which the reaction mixturewas warmed to room temperature and stirred for an additional 12 hours.The solvents were removed under reduced pressure, and the residue wasdissolved in about 100 ml CH₂ Cl₂, washed with about 75 ml water, 50 ml0.1 NHCl, and about 50 ml saturated NaCl. The organic layers were dried(MgSO₄) and filtered. The filtrate was evaporated under reduced pressureand the residue was flash chromatographed on silica gel (2.4×24 cm),eluting first with CH₂ Cl₂ and then with 1% methanol in CH₂ Cl₂.Fractions showing a single spot corresponding to the product were pooledand evaporated under reduced pressure to yield about 1.29 g of Compound34.

Diethyl N- 4-(aminomethyl)benzoyl!-L-glutamate (35)

About 1.8 ml trifluoroacetic acid was added dropwise to a stirredsolution containing about 1.0 g of Compound 34 in about 20 ml CH₂ Cl₂.The mixture was stirred at room temperature for 15 minutes, evaporatedto dryness under reduced pressure, and co-evaporated twice with about 30ml absolute ethanol. The residue was then subjected to columnchromatography on silica gel (1.5×15 cm), eluting with a gradient of5-10% methanol in CHCl₃ to yield about 0.68 g of Compound 35.

Diethyl N- 4- N-!(2,4-diaminofuro2,3-d!pyrimidin-5-yl)methyl(amino)methyl(benzoyl)-L-glutamate (37)

About 0.28 g of anhydrous K₂ CO₃ and about 0.67 g of Compound 35 wereadded to a solution containing about 0.2 g of Compound 35 in about 3 mlanhydrous DMSO. The reaction mixture was stirred under nitrogen at roomtemperature for 24 hours. The temperature was then raised to about 45°C. and the reaction continued for an additional 48 hours. The reactionmixture was then cooled to room temperature, diluted with about 50 mlwater and stirred for about 8 hours. The solid that separated wasfiltered, washed with water, air dried, and dissolved in methanol. About1 g of silica gel was added to the solution and the suspensionevaporated to dryness under reduced pressure. The silica plug was loadedon a dry silica gel column (2.4×17 cm) and eluted with a gradient of1-7% methanol in CHCl₃. Fractions corresponding to the product werepooled and evaporated to dryness. The residue was triturated with coldanhydrous ether to yield about 0.25 g of Compound 37.

N- 4- N-!(2,4-diaminofuro2,3-d!pyrimidin-5-yl)methyl!amino!methyl!benzoyl!-L-glutamic acid (34)

About 1 ml of 1N sodium hydroxide was added to a solution containingabout 0.1 g of Compound 37 in about 10 ml of 2:1 methanol/THF. Themixture was stirred at room temperature for about 24 hours. Thevolatiles were removed under reduced pressure, and the residue dissolvedin about 5 ml water and stirred for an additional 24 hours. The solutionwas cooled in an ice bath and the pH adjusted carefully to 4.0 bydropwise addition of 1N HCl. The precipitate was collected byfiltration, washed well with water and ether, and immediately driedunder high vacuum to yield about 0.08 g of Compound 34.

Example 13

Compounds 33 and 34 were evaluated as inhibitors of Lactobacillus caseiDHFR, human recombinant (REC) DHFR and DHFR isolated from human CCRF-CEMleukemic cells. The performance of these compounds was compared withthat of MTX. The compounds were also evaluated as inhibitors of L. caseiTS and human recombinant TS. The results are presented in Table 14below.

                  TABLE 14    ______________________________________    Inhibitory Concentrations (IC.sub.50 in μM)            Human             CCRF-            REC      L. casei CEM     Human  L. casei    Compound            DHFR     DHFR     DHFR    REC TS TS    ______________________________________    33      1.0      0.1      0.25    220     200    34      >200     >200     30.5    63.0   >200    MTX     0.004    0.006    0.0007  170    ND    ______________________________________

Example 14

Compounds 33 and 34 were also evaluated for their substrate activity inCCRF-CEM human leukemia folylpolyglutamate synthetase; the compoundswere compared against aminopterin. Results are presented in Table 15below.

                  TABLE 15    ______________________________________    Substrate Activity of Compounds 33 and 34 for CCRF-CEM Human    Leukemia Cell Folylpolyglutamate Synthetase    Substrate             K.sub.m, μM                         V.sub.max(rel)                                    V.sub.max /K.sub.m(rel)    ______________________________________    Aminopterin             4.8 ± 0.7 (N = 6)                         1 (N = 6)  0.21 ± 0.04 (N = 6)    33       8.5 ± 2.1 (N = 3)                         0.65 ± 0.01                                    0.07 ± 0.02 (N = 3)                         (N = 3)    34       ND          0.6 (N = 5)                                    ND    ______________________________________

Example 15

Compounds 33 and 34 were also tested for their growth inhibition ofhuman T-lymphoblastic leukemia cell line CCRF-CEM, its MTX-esistancesubline (R30dm), and human squamous cell carcinoma cell lines (FaDu andA235). The performance of these compounds was evaluated against MTX.Results are presented in Table 16 below.

                  TABLE 16    ______________________________________    Growth Inhibition (EC.sub.50, μM)    During Continuous Exposure (0 to 120 hours)    Com-    pound CCRF-CEM   R30DM      FaDu     A253    ______________________________________    33    0.29 ± 0.01                     4.25 ± 0.05                                0.018 ± 0.02                                         0.54 ±          (N = 3)    (N = 2)    (N = 2)  0.09 (N = 3)    34    48.0 ± 23.0                     ND         ND       ND          (N = 2)    MTX   0.014 ± 0.001                     0.018 ± 0.003                                0.017 ± 0.002                                         0.013 ±          (N= 10)    (N = 5)    (N = 5)  0.0008                                         (N = 3)    ______________________________________     Average values are presented ± range for N = 2 and ± standard     deviation for N ≧ 3.

Example 16

The following example provides methods for preparing the pyrido2,3-d!pyrimidine compounds and pharmaceutically acceptable salts thereofas described above. Reference letters and numerals correspond with thosein FIG. 13. Guanidine (FIG. 13b) is condensed with2-amino-3,5-dicarbonitrile-4-R₁₃ -pyridine (FIG. 13a), wherein R₁₃ is alower alkyl group having one to about seven carbon atoms as describedherein, in refluxing ethyl alcohol to produce 2,4-diaminopyrido2,3-d!pyrimidine-5-R₃ -6-carbonitrile (FIG. 13c). This product,2,4-diaminopyrido 2,3-d!pyrimidine-5-R₃ -6-carbonitrile (FIG. 13c), isthen subjected to reductive condensation with an alkyl amine, asubstituted aniline or benzylamine derivative containing the R₂ group asdescribed herein, such as for example, 3,4,5-trimethoxyaniline, andRaney nickel in aqueous acetic acid solution, and preferably about 70%acetic acid solution, to form 2,4-diamino-5-R₁₃ -6-(R₂)amino!methyl!pyrido 2,3-d!pyrimidine (FIG. 13d). The startingmaterial 2-amino-3,5-dicarbonitrile-4-R₃ -pyrimidine (FIG. 13a) may besynthesized by those skilled in the art by modifying the method ofPiper, et al., J. Med. Chem., Vol. 29, p. 1080 (1986).

These methods further include adding the product represented in FIG. 13dto about 37% formaldehyde in acetonitrile at about 25° C., adding sodiumcyanoborohydride, glacial acetic acid and methanol, and refrigeratingthe reaction mixture overnight to form 2,4-diamino-5-R₁₃ -6(R₂)methylamino!methyl!pyrido 2,3-d!pyrimidine (FIG. 13e).

2,4-Diamino-5-R₃ -6 (R₂)formylamino!methyl!pyrido 2,3-d!pyrimidine (FIG.13f) is prepared by reacting the product of FIG. 13d in about 98% formicacid as a solvent and acetic anhydride as a catalyst, removing thesolvent under reduced pressure, diluting the reaction product withmethanol and refrigerating the diluted reaction product overnight.

2,4-Diamino-5-R₃ -6 (R₂)nitrosoamino!methyl!pyrrido 2,3-d!pyrimidine(FIG. 13g) is prepared by reacting a chilled solution of the product ofFIG. 13d in aqueous acetic acid and dimethyl formamide (DMF) and thenadding NaNO₂ (sodium nitrate) in water. This mixture is stirred at about0° C. to 5° C. for about two hours and then poured into dilute-sodiumhydroxide.

It will be appreciated by those skilled in the art that by following thehereinbefore described methods of preparing the products of FIGS. 13d,13e, 13f and 13g of this invention that the derivatives of the productsof FIGS. 13d, 13e, 13f and 13g can be similarly prepared using theappropriate alkylamine, substituted aniline or benzylamine derivativecontaining the R₂ group as described herein.

Further, a method for preparing 4-amino-4-oxo derivatives of theproducts of FIGS. 13d, 13e, 13f or 13g of this invention includessubjecting these products to hydrolysis with 6N (six-normal solution)HCl for about six hours at room temperature.

Another embodiment of this invention is a method for preparing 2,4-dioxoderivatives of the products of FIGS. 13d, 13e, 13f or 13g that includessubjecting these products to hydrolysis with 6N HCl under mild refluxconditions for about two hours.

Example 17

The following example provides a method for preparing6-(thiophenylmethyl)-2,4-diaminopyrido 2,3-d!pyrimidine (FIG. 14e) and6-(thionapthylmethyl)-2,4-diaminopyrido 2,3-d!pyrimidine (FIG. 14f)generally represented by formula 11. Reference letters and numeralscorrespond with those in FIG. 14.

2,4-diaminopyrido 2,3-d!pyrimidine-6-carboxyaldehyde (FIG. 14b)

About 2.0 g of the nitrile (FIG. 14a) was dissolved in about 60 ml warmHCO₂ H under N₂. About 10 g of damp Raney Ni was added. The mixture wasrefluxed for 2 hours and filtered through Celite®. The filtrate wasconcentrated under reduced pressure at a temperature of 50° C. with theaid of EtOH. The resulting viscous orange residue was then dissolved inabout 150 ml of boiling H₂ O. The boiling solution was treated withNorit® and filtered through Celite® while hot. The filtrate wasneutralized to pH 7 with 1N NaOH to give a yellow precipitate. Thesuspension was refrigerated overnight, filtered and washed with H₂ O,EtOH and Et₂ O to yield about 1.75 g of a yellow solid. Examination byTLC (4:1:0.1 CHCl₃ :MeOH:NH₄ OH) showed a dominant UV-absorbing spot atR_(f) =0.38 and contamination spots at R_(f) =0.19 and at baseline. Thespot at R_(f) =0.19 was, after chromatographic separation, determined tocorrespond to the R_(f) value of compound (FIG. 14c).

2,4-diaminopyrido 2,3-d!pyrimidine-6-methanol (FIG. 14c)

About 5.0 g of crude aldehyde (FIG. 14b) was pulverized, dried andstirred in anhydrous MeOH under N₂ overnight. About 0.17 g of NaBH₄ wasadded to the mixture four times at intervals of 15 minutes. The mixturewas stirred for 5 additional hours. Insoluble material was filtered andthe filtrate was treated with about 200 ml H₂ O. The filtrate was thenconcentrated under reduced pressure at a temperature of 35° C. until ayellow precipitate began to form. The mixture was then refrigeratedovernight, filtered and rinsed with H₂ O, EtOH and Et₂ O to yield about1.50 g of a yellow solid. TLC (4:1:0.1 CHCL₃ :MeOH:NH₄ OH) showed aproduct spot at R_(f) =0.41 and a slight spot corresponding to thestarting material. Separation was carried out by chromatography withsilica gel.

6-(Bromomethyl)-2,4-diaminopyrido 2,3-d!pyrimidine (FIG. 14d)

About 0.24 g of crude alcohol (FIG. 14c) was dried with P₂ O₅ at 110° C.under vacuum overnight and then added to about 10 ml of anhydrousdioxane. The mixture was stirred in an ice bath while dry HBr gas wasbubbled through for 15 minutes, after which the flask was quicklystoppered. The mixture continued to stir and the alcohol dissolved afterapproximately 1/2 hour. The solution stirred for 24 hours and was thenadded dropwise to stirred Et₂ O under N₂ to give a yellow suspension.The suspension was refrigerated overnight, filtered and immediatelydried with P₂ O₅ under vacuum at 50° C. to yield about 45 mg of thecompound represented by FIG. 14d.

6-(Thiophenylmethyl-2,4-diaminopyrido 2,3-d!pyrimidine (FIG. 14e)

About 0.12 ml of phenylthiol was dissolved in about 10 ml DMAC and addeddropwise to about 0.25 g of the compound of FIG. 14d. About 1 g of K₂CO₃ was added to the mixture to adjust the pH to approximately 8. After1 hour, the compound of FIG. 14dwas not detectable by TLC (3:1:0.1 CHCl₃:MeOH:NH₄ OH). A product spot appeared at R_(f) =0.33 with contaminationspots at R_(f) =0.51 and at baseline. The solid was filtered and rinsedwith H₂ O, EtOH and Et₂ O to yield about 22 mg of the compound of FIG.14e.

6-(Thionaphthylmethyl)-2,4-diaminopyrido 2,3-d!pyrimidine (FIG. 14f)

About 0.07 g of napthylenethiol was dissolved in DMAC (15 mL) and addeddropwise to about 0.10 g of the compound of FIG. 14d. About 0.3 g of Na₂CO₃ was added and the color of the reaction mixture changed from yellowto green. The reaction was monitored by TLC (4:1:0.1 CHCl₃ :MeOH:NH₄OH). A product spot occurred at R_(f) =0.5. After 3 hours, startingmaterial was still present. Also, the yellow color returned. Thereaction was run overnight. The pH was then checked and found to beslightly acidic. The solution was added dropwise to about 100 ml of 1NNa₂ CO₃. The suspension was stirred for 15 minutes and refrigerated for4 hours. The solid was filtered and rinsed with H₂ O, EtOH and Et₂ O toyield about 25 mg of the compound of FIG. 14f

Example 18

The following are various methods for making various2,4-diamino-6-substituted-benzylamino pyrido 2,3-d!pyrimidines generallyrepresented by formula 11. Reference letters and numerals correspondwith those in FIG. 15. The synthesis of the desired compounds isachieved via the reductive amination of 2,4-diamino-6-amino pyrido2,3-d!pyrimidine 2, with the appropriately substituted aldehyde asgenerally illustrated in FIG. 15.

2,4-diamino-6-nitropyrido 2,3-d!pyrimidine (FIG. 15a)

About 1 equivalent of 2,4,6-triamino pyrimidine was suspended in about50 ml of refluxing absolute ethanol with stirring under an atmosphere ofnitrogen. Concentrated HCl was added dropwise to effect solution and assoon as solution occurred, about 1.2 equivalents of nitromalonaldehydewas added all at once. Within 10 minutes, a thick reddish voluminousprecipitate started forming. TLC analysis indicated the presence of ayellow spot corresponding to that of the desired product along withstaring materials. The reaction mixture was stirred at reflux for 3.5hours, immediately diluted with 30 ml of water, cooled and neutralizedwith concentrated NH₄ OH. The precipitate was collected on a funnel andwas washed repeatedly with water to remove unreacted triamino pyrimidineto yield pure 2,4-diamino-6-nitropyrido 2,3-d!pyrimidine.

Example 19

The following is a method of making 2,4-diamino-6-(anilinomethyl) pyrido2,3-d!pyrimidines generally represented by formula 11. Reference lettersand numerals correspond with those in FIG. 16.

About 1 equivalent of 2,4-diamino-pyrido 2,3-d!pyrimidine-6-carbonitrile(FIG. 16a) (achieved via literature procedures) was dissolved in 80%acetic acid. To this solution was added about 5 equivalents of RaneyNickel followed immediately by about 1.5 equivalents of theappropriately substituted aniline (FIG. 16b). The mixture washydrogenated under atmospheric pressure and at room temperature for 6hours. TLC analysis at the end of this period indicated the presence ofa spot corresponding to the desired product. The reaction mixture wasfiltered through Celite® and the filtrate was evaporated to dryness toyield a reddish residue. This residue was dissolved in warm absoluteethanol and then neutralized in the cold with 1N Na₂ CO₃ dropwise withstirring to deposit the crude product. This solid was collected byfiltration and was washed repeatedly with acetone and dissolved in alarge volume of methanol; silica gel was added and the methanol strippedoff to yield a dry plug. Column chromatography using CHCl₃ :MeOH (5:1)as the eluant yielded pure target compounds represented by FIG. 16c.

Example 20

The following example provides methods for making pyrido3,2-d!pyrimidine compounds generally represented by formula 11.Reference letters and numerals correspond with those of FIG. 17.

2,4-dioxo-6-methylpyrido 3,2-d!pyrimidine (FIG. 17a)

About 20 g of 5-aminouracil, 80 ml of 20% HCl and 4 ml of crotonaldehydewere heated together under reflux for 1 hour. The solution wasevaporated to dryness under rotary evaporation. Water was added to theresidue so as to make the mixture just stirrable and then it wastriturated with ammonium hydroxide with strong stirring until the pHincreased to 10-11. Stirring was continued for another 10 minutes. Theprecipitate was filtered and was washed with minimal methanol and thenchloroform and dried to yield about 17.58 g of the compound of FIG. 17a.

6-(Acetoxymethyl)-2,4-dioxopyrido 3,2-dopyrimidine (FIG. 17b)

About 1.77 g of the compound of FIG. 17a in 50 ml of glacial acetic acidcontaining about 6.5 g of MCPBA (57-85%) was refluxed for 3 hours. About40 ml of acetic anhydride was added to the hot reaction mixture and therefluxing was continued for another 30 minutes. The clear brown solutionwas evaporated to dryness and the solid was stirred with about 100 mlether and filtered. The solid was crystallized from ethanol to yieldabout 1.55 g of the compound of FIG. 17b.

6-(Acetoxymethyl)-2,4-dichloropyrido 3,2-d!pyrimidine (FIG. 17c)

About 1.5 g of the compound of FIG. 17c was refluxed with about 38 mL ofphosphoryl chloride containing about 2.5 mL of triethylamine for 8hours. The volume was reduced to about 5 mL by rotary evaporation. Thedark syrup which resulted was poured into crushed ice. The coldsuspension was extracted with methylene chloride (3×50 mL) and washedwith cold water until the washing were neutral. The organic layer wasdried over anhydrous sodium sulfate and evaporated to dryness underrotary evaporation. The dark solid residue was stirred and refluxed withpetroleum ether (30°-60° C.) and a suitable amount of decoloringcharcoal, and filtered through Celite®, which was repeated twice. Thecombined liquid solution was concentrated until the light yellow solidprecipitated out and was allowed to cool to room temperature and storedin a refrigerator for 2 hours. The crystallized solid was filtered anddried to yield about 0.86 g of the compound of FIG. 17c.

2,4-diamino-6-(hydroxymethyl)pyrido 3,2-d!pyrimidine (FIG. 17d)

About 2.5 g of the compound of FIG. 17c was heated with 30 mL of liquidammonia in a sealed bomb at between about 150°-170° C. for 18 hours.After cooling to room temperature, the bomb was opened and the liquidammonia was allowed to evaporate at room temperature. The solid wascrystallized from glacial acetic acid and a small amount of water toyield about 1.24 g of the compound of FIG. 17d.

2,4-diamino-6-(bromomethyl)pyrido 3,2-d!pyrimidine (FIG. 17e)

A suspension of about 0.72 g of the compound of FIG. 17d in 12 mL dryTHF was stirred for 8 hours with 1 mL of phosphorus tribromide. Theprecipitated solid was filtered, washed with cold 50% THF-Ether, anddried to give the compound of FIG. 17e. Because of the instability, thiscompound was not purified further. The ¹ HNMR showed that the majorityof the solid was the desired compound.

2,4-diamino-6-(paramethoxyanilinylmethyl)pyrido 3,2-d!pyrimidine (FIG.17f)

To a suspension of about 3.5 mmol of the compound of FIG. 17e inanhydrous dimethylacetamide was added 0.92 g anisidine and 1.03 ganhydrous potassium. After the suspension was stirred for 2 days, almostall of the compound of FIG. 17e disappeared. The solvent DMAC wasremoved under diminished pressure. The solid residue was washed withmethanol three times and filtered. To the combined liquid was addedsilica gel and the methanol was evaporated to dryness. Separation toafford pure product of FIG. 17f was carried out by columnchromatography.

2,4-diamino-6-(phenylthiomethyl)pyrido 3,2-d!pyrimidine (FIG. 17g)

To a suspension of about 2.5 mmol of the compound of FIG. 17g inanhydrous dimethylacetamide was added 1 mL thiophenol and 690 mganhydrous potassium. The suspension was stirred for 3 days. The solventDMAC was removed under diminished pressure. The solid residue was washedwith methanol three times and filtered. The combined liquid was added tosilica gel and then the methanol was evaporated to dryness. A smallamount of product was separated through a dry column. Aftercrystallization, about 5 mg of pure product of FIG. 17g was obtained.

2,4-diamino-6-(naphathalinylmethyl)pyrido 3,2-d!pyrimidine (FIG. 17h)

The procedure for making the compound of FIG. 17f was repeated with areaction time of 5 days to yield the compound of FIG. 17h.

Example 21

The following example provides methods for making the tricyclicpyrimidine compounds generally represented by formula 10. Referenceletters and numerals correspond with those of FIG. 10.

N-butoxycarbonyl-4-piperidone (FIG. 10b)

About 2 g of 4-piperidone hydrochloride monohydrate (FIG. 10a) wasdissolved in about 30 ml of N,N-dimethylformamide at temperatures ofbetween about 110°-115° C. The soultion was cooled to room termperature,and to this solution was added about 2.6 g of triethyl amine and asolution containing about 3.06 g of ditertiarybutyl dicarbonate in 10 mlDMF. The reaction was continued for about 24 hours at room temperature.The DMF was removed under reduced pressure. About 100 ml of water wasadded to the residue and the mixture was extracted with ethyl ether(2×100 ml), and the organic layer dried over anhydrous MgSO₄. The etherwas evaporated to dryness under reduced pressure to yield about 2.33 gof the compound of FIG. 10b.

3-bromo-4-piperidone hydrobromide (FIG. 10c)

About 2.6 g of N-butoxycarbonyl-4-piperdone (FIG. 10b) was dissolved inabout 70 ml of chloroform; to this solution was added about 2.08 g Br₂over a period of about 30 minutes. The reaction was continued for about2 hours at room temperature during which a white precipitate of thecompound of FIG. 10c was formed. The reaction mixture was filtered andwashed with chloroform ether to yield about 2.76 g of the compound ofFIG. 10c.

2,4-diamino-5,6,7,8-tetrahydro-7-pyrido 4',3':4,5 furo 2,3-dipyrimidinehydrobromide (FIG. 10f)

A solution containing about 1.83 g of the compound of FIG. 10c in about10 ml of anhydrous DMF was added dropwise to a suspension containingabout 0.504 g of 2,4-diamino-6-hydroxypyrimidine in about 3 ml ofanhydrous DMF. The reaction became a clear solution after about 1 hour;the soultion was then left at room temperature for about 48 hours. Thewhite precipitate which formed was collected by filitration and airdried to yield about 0.66 g of the compound of FIG. 10f.

2,4-diamino-5,6,7,8-tetrahydro-(7-butoxycarbonyl)pyrido 4',3':4,5!furo2,3-d!pyrimidine hydrobromide (FIG. 10e)

The filtrate obtained in preparing the compound of FIG. 10c was dilutedwith about 150 ml of chloroform and washed with water, saturated sodiumbicarbonate and brine. The organic layer was dried over anhydrous MgSO₄,and the chloroform was removed under reduced pressure to yield a viscousbrown oil. The residue was dissolved immediately in about 5 ml ofanhydrous DMF, and added to a suspension containing about 0.252 g of2,4-diamino-6-hydroxypyrimidine in anhydrous DMF. The reaction wascontinued for about 48 hours at room temperature. The DMF was removedunder reduced presure, and the residue was dissolved in 50 ml ofmethanol; about 1.7 g of silica gel was added and the mixture wasevaporated to dryness under reduced pressure. About 50 ml of ether wasadded to the silica gel plug; the homogenous plug collected afterfiltration was air-dried and then placed on top of a dry silica gelcolumn (1.5 cm×10 cm) and gradiantly eluted with MeOH in CHCl₃.Fractions containing the compound of FIG. 10e were pooled and evaporatedto dryness under reduced pressure to yield 0.025 g of the compound.

2,4-diamino-5,6,7,8-tetrahydro-7-(benzyl)pyrido 4',3':4,5!furo2,3-d!pyrimidine (FIG. 10g)

About 0.46 g of the compound of FIG. 10f was suspended in 5 ml ofanhydrous DMSO. About 0.483 g of anhydrous potassium carbonate and 0.24g of benzyl bromide were added to the suspension; the reaction wascontinued for 24 hours at room temperature. The mixture was then dilutedwith about 30 ml of water and stirred for 24 hours at room temperature.The resulting precipitate was collected by filtration, washed withwater, acetone, ether and air-dried. The crude solid was dissolved in amixture of DMF:MeOH (1:5); about 1.2 g of silica gel was added and themixture was evaporated to dryness under reduced pressure. The resultingsilica gel plug was placed on a top of a dry silica gel column (1.5cm×10 cm) and gradiantly eluted with MeOH in CHCl₃. The fractionscontaining the compound of FIG. 10g were pooled and evaporated todryness under reduced pressure; the resulting solid was triturated withether and filtered to yield about 0.156 g of product.

2,4-diamino-5,6,7,8-tetrahydro-7- (3',4',5'-trimethoxy)benzyl!pyrido4',3':4,5!furo 2,3-d!pyrimide (FIG. 10h)

The compound of FIG. 10h was prepared and purified in the same manner asthe compound of FIG. 10g, only using 3',4',5'-trimethoxybenzyl chlorideinstead of benzyl bromide to yield about 0.12 g of the compound of FIG.10h as a yellow solid.

2,4-diamino-5,6,7,8-tetrahydro-7- (3',5'-dimethoxy)benzyl!pyrido4',3':4,5!furo 2,3-d!pyrimide (FIG. 10i)

The compound of FIG. 10i was prepared and purified in the same manner asthe compound of FIG. 10g, only using 3',5'-dimethoxybenzyl chlorideinstead of benzyl bromide to yield about 0.126 g of the compound of FIG.10i.

2,4-diamino-5,6,7,8-tetrahydro-7- (2',4'-dichloro)benzyl!pyrido4',3':4,5!furo 2,3-d!pyrimide (FIG. 10j)

The compound of FIG. 10j was prepared and purified in the same manner asthe compound of FIG. 10g, only using 2',4'-dichlorobenzyl chlorideinstead of benzyl bromide to yield about 0.151 g of the compound of FIG.10j.

2,4-diamino-5,6,7,8-tetrahydro-7- (3',4'-dichloro)benzyl!pyrido4',3':4,5!furo 2,3-d!pyrimide (FIG. 10k)

The compound of FIG. 10k was prepared and purified in the same manner asthe compound of FIG. 10g, only using 3',4'-dichlorobenzyl chlorideinstead of benzyl bromide to yield about 0.131 g of the compound of FIG.10k.

2,4-diamino-5,6,7,8-tetrahydro-7- (2',6'-dichloro)benzyl!pyrido4',3':4,5!furo 2,3-d!pyrimide (FIG. 10l)

The compound of FIG. 10l was prepared and purified in the same manner asthe compound of FIG. 10g, only using 2',6'-dichlorobenzyl chlorideinstead of benzyl bromide to yield about 0.093 g of the compound of FIG.10l.

2,4-diamino-5,6,7,8-tetrahydro-7- (2',4'-dichloro)benzyl!pyrido4',3':4,5!furo 2,3-d!pyrimide (FIG. 10m)

2,4-Diamino-5,6,7,8-tetrahydro-7- (4"-benzoyl)diethyl-L-glutamicacid)!pyrido 4',3':4,5!furo 2,3-d!pyrimidine was prepared and purifiedin the same manner as the compound of FIG. 10g, only using4'-(chloromethyl)benzoyl glutamic acid diethyl ester instead of benzylbromide. About 1.5 ml of 1N NaOH was added to a solution containingabout 0.183 g of this compound in 10 ml methoxyethanol, and the solutionstirred at room temperature for about 24 hours. The ethoxyethanol wasevaporated under reduced presure, the residue was dissolved in about 10ml of water and stirring continued for an additional 24 hours. Thesolution was cooled in an ice bath and the pH was adusted to about 3.5via dropwise addition of 1N HCl. The precipitate formed was collected byfiltration, washed with water, acetone and ether and air-dried to obtainabout 0.160 g of the compound of FIG. 10m.

Example 22

The compound of FIG. 10m was tested for DHFR inhibition, as discussedabove in Example 1, against Pneumocystis carinii (Pc), Toxoplasmosisgondii (Tg), Macrobacterium avium (Ma) and rat liver (RL). Ma is anopportunistic infection in HIV infected patients. Table 17 sets forththe IC₅₀ values.

                  TABLE 17    ______________________________________    Inhibitory Concentrations (IC.sub.50, microM) and Selectivity Ratios    Compound Pc     Tg     RL   RL/Pc  RL/Tg M. avium    ______________________________________    10m      10.9   21.5   85.8 7.9    4     0.97    ______________________________________

The compound FIG. 10m showed a weak inhibitory activity against DHFR,but displayed promising selectivity ratios of 7.9 and 4 against Pc DHFRand Tg DHFR, respectively, against RL DHFR. In addition, compound 10mshowed significat inhibitory activity against Ma DHFR and had aselectivity ratio of 88 versus RL DHFR.

In addition, the ability of the compound of FIG. 10m to function as asubstrate of human CCRF-CEM folylpolyglutamate synthetase was assessed,as discussed sbove in Example 4. Results are presented in Table 18.

                  TABLE 18    ______________________________________    Activity of the Compound of FIG. 10m as Substrate for    CCRF-CEM Human Leukemia Cell Folylpolyglutamate Synthetase    substrate             K.sub.m, microM                        V.sub.max,rel                                   V.sub.max /K.sub.m(rel)                                            n    ______________________________________    Aminopterin             4.8 ± 0.7                        1          0.21 ± 0.04                                            6    10m      6.2 ± 1.4                        0.29 ± 0.05                                   0.06 ± 0.01                                            2    ______________________________________

Example 23

The following example provides methods for making pyrido3,2-d!pyrimidine compounds generally represented by formula 11.Reference letters and numerals correspond with those of FIG. 11.

The compound of FIG. 11a was synthesized from 2,6-dichloropyridine intwo steps--nitration at the 3 position with concentrated sulfuric acidand 90% nitric acid, followed by substitution of the 2-chloro moietywith a cyano group using CuCN at 180° C. The compound of FIG. 11b wassynthesized via a direct aromatic substitution of FIG. 11a witharylamines, 3',4',5'-trimethoxybenzylamine, or N-methylarylamines in 2ethoxyethanol at temperatures between about 120°-140° C. The compound ofFIG. 11b was then reduced by iron in acidic conditions under reflux inmethanol to yield the intermediate 2-cyano-3-amino-6-substitutedpyridines (FIG. 11c). Monomethylglycol was added to increase thesolubility. The compound of FIG. 11c was condensed with formamidinehydrochloride in dimethyl sulfone at temperatures between about 120° and150° C. to provide the compound of FIG. 11d.

Example 24

The following example provides methods for making pyrido3,2-d!pyrimidine compounds generally represented by formula 11.Reference letters and numerals correspond with those of FIG. 12.

The compound of FIG. 12a was reduced with iron powder, followed bycondensation with chlorohydrochloride in DMSO at temperatures of betweenabout 120° and 150° C. to yield 2,4-diamino-6-chloro-pyrido3,2-d!pyrimidine (FIG. 12c). The compound of FIG. 12c was subjected to asubstitution reaction with substituted thiophenols, to yield threearylthiol compounds (FIGS. 12f, 12g, and 12h) generally represented byFIG. 12d. The oxidation of these compounds with hydrogen peroxide inacetic acid provided three corresponding sulfonyl compounds with thegeneral structure represented by FIG. 12e.

Example 25

The following example provides methods for making pyrido3,2-d!pyrimidine compounds generally represented by formula 11.Reference letters and numerals correspond with those of FIG. 18.

The compound of FIG. 18a was prepared from 2,6-dichloropyridine bymethods known in the art. The 3-amino group of the compound of FIG. 18awas protected with an acetyl group by reacting the compound with acetylanhydride and triethylamine in the presence of DMAP under refluxconditions in methylene chloride for 20 hours. Part of the startingmaterial was converted to the N,N-diacetylated amino derivative, whichwas in turn converted in situ into the compound of FIG. 18b when stirredin a saturated sodium bicarbonate solution at room temperature for about1.5 hours. The compound of FIG. 18b was then oxidated withtrifluroperacetic acid in methylene chloride at room temperature for 24hours to yield the compound of FIG. 18c, 2-amidylpyridine-N-oxide. Thecompound of FIG. 18c was then heated at about 140° C. with a guanidinecarbonate base in 2-ethoxyethanol for about one hour to yield thecompound of FIG. 18d, 6-chloro-2-methyl-4-oxo-pyrido 3,2-d!pyrimidine.The compound of FIG. 18d was then reacted with the sodium salt ofp-thiocresol in anhydrous DMF at 125° C. to yield the pyrido3,2-d!pyrimidine compound of FIG. 18e.

Any compatible sulfur, oxygen or nitrogen containing nucleophile can bereacted with the compound of FIG. 18d to yield the corresponding pyrido3,2-d!pyrimidine compound.

It will be appreciated by those skilled in the art that the presentinvention provides compounds, and pharmaceutically acceptable saltsthereof, effective against infections caused by Pneumocystis carinii andToxoplasmosis gondii, methods of preparing these compounds, and methodsof using these compounds in a patient for therapeutic or prophylacticpurposes. It will be further appreciated by those skilled in the artthat this invention provides compounds, and pharmaceutically acceptablesalts thereof, effective in reducing tumors or otherwise destroyingcancerous cells in patients with cancer, methods of preparing thesecompounds, and methods of using these compounds in a patient fortherapeutic purposes.

Whereas particular embodiments of this invention have been describedabove for purposes of illustration, it will be evident to those skilledin the art that numerous variations of the details of the presentinvention may be made without departing from the invention as defined inthe appended claims.

What is claimed is:
 1. A compound, and pharmaceutically acceptable saltsthereof, having the formula: ##STR68## wherein X and Y are the same ordifferent and are selected from the group consisting of OH, NH₂, H andCH₃ ;wherein L and M are selected from the group consisting of carbonand CH, the chemical bond between L and M is selected from the groupconsisting of a single bond and a double bond, L and M are carbon whenthe bond is a double bond, and L and M are CH when the bond is a singlebond; wherein Z₂ and Z₃ are different and are selected from the groupconsisting of R₄ and ##STR69## where Z₂ is R₄ when Z₃ is ##STR70## andZ₂ is ##STR71## when Z₃ is R₄ ; wherein A is selected from the groupconsisting of CH and zero;wherein B is selected from the groupconsisting of CH, nitrogen, N--CH₂, CH₂ --N, CH₂ --CH₂, oxygen, sulfur,sulfoxide, sulfone and zero, but B is not N--CH₂ when A is CH and Z₃ isR₄ ; wherein A is not zero when B is CH₂ --CH₂ and Z₃ is R₄ ; wherein R₁is selected from the group consisting of hydrogen, a lower alkyl group,a nitroso group, a formyl group and zero and R₁ is zero when B is zero,oxygen, sulfur, sulfoxide or sulfone; wherein R₃ is selected from thegroup consisting of hydrogen, a lower alkyl group and zero when Z₂ isR₄, and R₃ is selected from the group consisting of a lower alkyl groupand zero when Z₃ is R₄, and R₃ is zero when A is zero; wherein R₄ isselected from the group consisting of hydrogen and a lower alkyl group;wherein R₅ is selected from the group consisting of hydrogen and a loweralkyl group; wherein R₈ is selected from the group consisting ofnaphthyl, mono-, di- and tri-substituted naphthyl, thionaphthyl,thiophenyl and hydroxyphenyl when R₁ is hydrogen and R₄ is hydrogen;wherein R₈ is selected from the group consisting of phenyl, mono-, di-and tri-substituted phenyl, naphthyl, mono-, di- and tri-substitutednaphthyl, pyridine and p-aroyl-L-glutamate when R₁ is a lower alkylgroup and R₄ is hydrogen; wherein R₈ is selected from the groupconsisting of pyridine, phenyl, mono-, di- and tri-substituted phenyl,naphthyl, and mono-, di- and tri-substituted naphthyl andp-aroyl-L-glutamate when R₁ is zero; wherein R₈ is selected from thegroup consisting of phenyl, mono-, di- and tri-substituted phenyl,naphthyl, mono-, di- and tri-substituted naphthyl andp-aroyl-L-glutamate when R₁ is hydrogen and R₄ is a lower alkyl group;and wherein R₈ is not p-benzoyl-L-glutamate or pyridine when X is OH, Ais zero, B is sulfur, R₄ is methyl and R₅ is hydrogen, and R₈ is notp-benzoyl-L-glutamate when X is OH, A is CH, B is CH, R₄ is hydrogen andR₅ is hydrogen; and wherein each lower alkyl group is independentlyselected from the group consisting of lower alkyl groups having from 1to 6 carbons.
 2. A method of therapeutically treating a patient for anillness comprising the steps of:a) employing a compound, orpharmaceutically acceptable salts thereof, having the formula: ##STR72##wherein X and Y are the same or different and are selected from thegroup consisting of OH, NH₂, H and CH₃ ;wherein L and M are selectedfrom the group consisting of carbon and CH, the chemical bond between Land M is selected from the group consisting of a single bond and adouble bond, L and M are carbon when the bond is a double bond, and Land M are CH when the bond is a single bond; wherein Z₂ and Z₃ aredifferent and are selected from the group consisting of R₄ and ##STR73##where Z₂ is R₄ when Z₃ is ##STR74## and Z₂ is ##STR75## when Z₃ is R₄ ;wherein A is selected from the group consisting of CH and zero;wherein Bis selected from the group consisting of CH, nitrogen, N--CH₂, CH₂ --N,CH₂ --CH₂, oxygen, sulfur, sulfoxide, sulfone and zero, but B is notN--CH₂ when A is CH and Z₃ is R₄ ; wherein A is not zero when B is CH₂--CH₂ and Z₃ is R₄ ; wherein R₁ is selected from the group consisting ofhydrogen, a lower alkyl group, a nitroso group, a formyl group and zeroand R₁ is zero when B is zero, oxygen, sulfur, sulfoxide or sulfone;wherein R₃ is selected from the group consisting of hydrogen, a loweralkyl group and zero when Z₂ is R₄ and R₃ is selected from the groupconsisting of a lower alkyl group and zero when Z₃ is R₄, and R₃ is zerowhen A is zero; wherein R₄ is selected from the group consisting ofhydrogen and a lower alkyl group; wherein R₅ is selected from the groupconsisting of hydrogen and a lower alkyl group; wherein R₈ is selectedfrom the group consisting of naphthyl, mono-, di- and tri-substitutednaphthyl, thionaphthyl, thiophenyl and hydroxyphenyl when R₁ is hydrogenand R₄ is hydrogen; wherein R₈ is selected from the group consisting ofphenyl, mono-, di- and tri-substituted phenyl, naphthyl, mono-, di- andtri-substituted naphthyl, pyridine and p-aroyl-L-glutamate when R₁ is alower alkyl group and R₄ is hydrogen; wherein R₈ is selected from thegroup consisting of pyridine, phenyl, mono-, di- and tri-substitutedphenyl, naphthyl, mono-, di- and tri-substituted naphthyl andp-aroyl-L-glutamate when R₁ is zero; wherein R₈ is selected from thegroup consisting of phenyl, mono-, di- and tri-substituted phenyl,naphthyl, mono-, di- and tri-substituted naphthyl andp-aroyl-L-glutamate when R₁ is hydrogen and R₄ is a lower alkyl group;wherein R₈ is not p-benzoyl-L-glutamate or pyridine when X is OH, A iszero, B is sulfur, R₄ is methyl and R₅ is hydrogen, and R₈ is notp-benzoyl-L-glutamate when X is OH, A is CH, B is CH, R₄ is hydrogen andR₅ is hydrogen; and wherein each lower alkyl group is independentlyselected from the group consisting of lower alkyl groups having from 1to 6 carbons; b) incorporating said compound in a suitablepharmaceutical carrier; and c) administering a therapeutically effectiveamount of said compound incorporated in said carrier to a patient. 3.The method of claim 2, wherein said illness is cancer.
 4. The method ofclaim 2, wherein said illness is selected from the group consisting ofinfection caused by Pneumocystis carinii and Toxoplasmosis gondii. 5.The method of claim 2, wherein said carrier is selected from the groupconsisting of physiologic saline and 5% dextrose for injection.
 6. Themethod of claim 2, including administering said compound incorporated insaid carrier to a patient parenterally.
 7. The method of claim 2,including administering said compound incorporated in said carrier to apatient orally.
 8. The method of claim 2, including administering saidcompound incorporated in said carrier to a patient topically.
 9. Amethod of prophylactically treating a patient for an illness comprisingthe steps of:a) employing a compound, or pharmaceutically acceptablesalts thereof, having the formula: ##STR76## wherein X and Y are thesame or different and are selected from the group consisting of OH, NH₂,H and CH₃ ;wherein L and M are selected from the group consisting ofcarbon and CH, the chemical bond between L and M is selected from thegroup consisting of a single bond and a double bond, L and M are carbonwhen the bond is a double bond, and L and M are CH when the bond is asingle bond; wherein Z₂ and Z₃ are different and are selected from thegroup consisting of R₄ and ##STR77## where Z₂ is R₄ when Z₃ is ##STR78##and Z₂ is ##STR79## when Z₃ is R₄ ; wherein A is selected from the groupconsisting of CH and zero;wherein B is selected from the groupconsisting of CH, nitrogen, N--CH₂, CH₂ --N, CH₂ --CH₂, oxygen, sulfur,sulfoxide, sulfone and zero, but B is not N--CH₂, when A is CH and Z₃ isR₄ ; wherein A is not zero when B is CH₂ --CH₂ and Z₃ is R₄ ; wherein R₁is selected from the group consisting of hydrogen, a lower alkyl group,a nitroso group, a formyl group and zero and R₁ is zero when B is zero,oxygen, sulfur, sulfoxide or sulfone; wherein R₃ is selected from thegroup consisting of hydrogen, a lower alkyl group and zero when Z₂ is R₄and R₃ is selected from the group consisting of a lower alkyl group andzero when Z₃ is R₄, and R₃ is zero when A is zero; wherein R₄ isselected from the group consisting of hydrogen and a lower alkyl group;wherein R₅ is selected from the group consisting of hydrogen and a loweralkyl group; wherein R₈ is selected from the group consisting ofnaphthyl, mono-, di- and tri-substituted naphthyl, thionaphthyl,thiophenyl and hydroxyphenyl when R₁ is hydrogen and R₄ is hydrogen;wherein R₈ is selected from the group consisting of phenyl, mono-, di-and tri-substituted phenyl, naphthyl, mono-, di- and tri-substitutednaphthyl, pyridine and p-aroyl-L-glutamate when R₁ is a lower alkylgroup and R₄ is hydrogen; wherein R₈ is selected from the groupconsisting of pyridine, phenyl, mono-, di- and tri-substituted phenyl,naphthyl, mono-, di- and tri-substituted naphthyl andp-aroyl-L-glutamate when R₁ is zero; wherein R₈ is selected from thegroup consisting of phenyl, mono-, di- and tri-substituted phenyl,naphthyl, mono-, di- and tri-substituted naphthyl andp-aroyl-L-glutamate when R₁ is hydrogen and R₄ is a lower alkyl group;wherein R₈ is not p-benzoyl-L-glutamate or pyridine when X is OH, A iszero, B is sulfur, R₄ is methyl and R₅ is hydrogen, and R₈ is notp-benzoyl-L-glutamate when X is OH, A is CH, B is CH, R₄ is hydrogen andR₅ is hydrogen; and wherein each lower alkyl group is independentlyselected from the group consisting of lower alkyl groups having from 1to 6 carbons; b) incorporating said compound in a suitablepharmaceutical carrier; and c) administering a prophylacticallyeffective amount of said compound incorporated in said carrier to apatient; wherein said illness is selected from the group consisting ofcancer and infection caused by Pneumocystis carinii and Toxoplasmosisgondii.
 10. The method of claim 9, wherein said carrier is selected fromthe group consisting of physiologic saline and 5% dextrose forinjection.
 11. The method of claim 9, including administering saidcompound incorporated in said carrier to a patient parenterally.
 12. Themethod of claim 9, including administering said compound incorporated insaid carrier to a patient orally.
 13. The method of claim 9, includingadministering said compound incorporated in said carrier to a patienttopically.